Modified RNAi agents

ABSTRACT

One aspect of the present invention relates to double-stranded RNAi (dsRNA) duplex agent capable of inhibiting the expression of a target gene. The dsRNA duplex comprises one or more motifs of three identical modifications on three consecutive nucleotides in one or both strand, particularly at or near the cleavage site of the strand. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.

RELATED APPLICATION

This application claims priority to PCT Application No.PCT/US2012/065601, filed Nov. 18, 2012, which claims benefit of priorityto U.S. Provisional Application No. 61/561,710, filed on Nov. 18, 2011;the entire contents of which are hereby incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates to RNAi duplex agents having particular motifsthat are advantageous for inhibition of target gene expression, as wellas RNAi compositions suitable for therapeutic use. Additionally, theinvention provides methods of inhibiting the expression of a target geneby administering these RNAi duplex agents, e.g., for the treatment ofvarious diseases.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNAi (dsRNA)can block gene expression (Fire et al. (1998) Nature 391, 806-811;Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directsgene-specific, post-transcriptional silencing in many organisms,including vertebrates, and has provided a new tool for studying genefunction. RNAi is mediated by RNA-induced silencing complex (RISC), asequence-specific, multi-component nuclease that destroys messenger RNAshomologous to the silencing trigger. RISC is known to contain short RNAs(approximately 22 nucleotides) derived from the double-stranded RNAtrigger, but the protein components of this activity remained unknown.

Double-stranded RNA (dsRNA) molecules with good gene-silencingproperties are needed for drug development based on RNA interference(RNAi). An initial step in RNAi is the activation of the RNA inducedsilencing complex (RISC), which requires degradation of the sense strandof the dsRNA duplex. Sense strand was known to act as the first RISCsubstrate that is cleaved by Argonaute 2 in the middle of the duplexregion. Immediately after the cleaved 5′-end and 3′-end fragments of thesense strand are removed from the endonuclease Ago2, the RISC becomesactivated by the antisense strand (Rand et al. (2005) Cell 123, 621).

It was believed that when the cleavage of the sense strand is inhibited,the endonucleolytic cleavage of target mRNA is impaired (Leuschner etal. (2006) EMBO Rep., 7, 314; Rand et al. (2005) Cell 123, 621; Schwarzet al. (2004) Curr. Biol. 14, 787). Leuschner et al. showed thatincorporation of a 2′-O-Me ribose to the Ago2 cleavage site in the sensestrand inhibits RNAi in HeLa cells (Leuschner et al. (2006) EMBO Rep.,7, 314). A similar effect was observed with phosphorothioatemodifications, showing that cleavage of the sense strand was requiredfor efficient RNAi also in mammals.

Morrissey et al. used a siRNA duplex containing 2′-F modified residues,among other sites and modifications, also at the Ago2 cleavage site, andobtained compatible silencing compared to the unmodified siRNAs(Morrissey et al. (2005) Hepatology 41, 1349). However, Morrissey'smodification is not motif specific, e.g., one modification includes 2′-Fmodifications on all pyrimidines on both sense and antisense strands aslong as pyrimidine residue is present, without any selectivity; andhence it is uncertain, based on these teachings, if specific motifmodification at the cleavage site of sense strand can have any actualeffect on gene silencing activity.

Muhonen et al. used a siRNA duplex containing two 2′-F modified residuesat the Ago2 cleavage site on the sense or antisense strand and found itwas tolerated (Muhonen et al. (2007) Chemistry & Biodiversity 4,858-873). However, Muhonen's modification is also sequence specific,e.g., for each particular strand, Muhonen only modifies either allpyrimidines or all purines, without any selectivity.

Choung et al. used a siRNA duplex containing alternative modificationsby 2′-OMe or various combinations of 2′-F, 2′-OMe and phosphorothioatemodifications to stabilize siRNA in serum to Sur10058 (Choung et al.(2006) Biochemical and Biophysical Research Communications 342,919-927). Choung suggested that the residues at the cleavage site of theantisense strand should not be modified with 2′-OMe in order to increasethe stability of the siRNA.

There is thus an ongoing need for iRNA duplex agents to improve the genesilencing efficacy of siRNA gene therapeutics. This invention isdirected to that need.

SUMMARY

This invention provides effective nucleotide or chemical motifs fordsRNA agents optionally conjugated to at least one ligand, which areadvantageous for inhibition of target gene expression, as well as RNAicompositions suitable for therapeutic use.

The inventors surprisingly discovered that introducing one or moremotifs of three identical modifications on three consecutive nucleotidesat or near the cleavage site of a dsRNA agent that is comprised ofmodified sense and antisense strands enhances the gene silencingactivity of the dsRNA agent.

In one aspect, the invention relates to a double-stranded RNAi (dsRNA)agent capable of inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 30 nucleotides. The dsRNA duplex is represented by formula(III):

(III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′ n_(p)′-N_(a)′-(X′ X′ X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)₁-N_(a)′-n_(q)′ 5′,In formula (III), i, j, k, and l are each independently 0 or 1; p and qare each independently 0-6; n represents a nucleotide; each N_(a) andN_(a)′ independently represents an oligonucleotide sequence comprising0-25 nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; each n_(p) and n_(q)independently represents an overhang nucleotide sequence comprising 0-6nucleotides; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ eachindependently represent one motif of three identical modifications onthree consecutive nucleotides; wherein the modifications on Nb isdifferent than the modification on Y and the modifications on Nb′ isdifferent than the modification on Y′. At least one of the Y nucleotidesforms a base pair with its complementary Y′ nucleotides, and wherein themodification on the Y nucleotide is different than the modification onthe Y′ nucleotide.

Each n_(p) and n_(q) independently represents an overhang nucleotidesequence comprising 0-6 nucleotides; each n and n′ represents anoverhang nucleotide; and p and q are each independently 0-6.

In another aspect, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 30nucleotides. The sense strand contains at least two motifs of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site within the strandand at least one of the motifs occurs at another portion of the strandthat is separated from the motif at the cleavage site by at least onenucleotide. The antisense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site within the strandand at least one of the motifs occurs at another portion of the strandthat is separated from the motif at or near cleavage site by at leastone nucleotide. The modification in the motif occurring at or near thecleavage site in the sense strand is different than the modification inthe motif occurring at or near the cleavage site in the antisensestrand.

In another aspect, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 30nucleotides. The sense strand contains at least one motif of three 2′-Fmodifications on three consecutive nucleotides, where at least one ofthe motifs occurs at or near the cleavage site in the strand. Theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at or near the cleavagesite.

In another aspect, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 30nucleotides. The sense strand contains at least one motif of three 2′-Fmodifications on three consecutive nucleotides at positions 9,10,11 fromthe 5′ end. The antisense strand contains at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at positions11,12,13 from the 5′ end.

In another aspect, the invention further provides a method fordelivering the dsRNA to a specific target in a subject by subcutaneousor intravenenuous administration.

DETAILED DESCRIPTION

A superior result may be obtained by introducing one or more motifs ofthree identical modifications on three consecutive nucleotides into asense strand and/or antisense strand of a dsRNA agent, particularly ator near the cleavage site. The sense strand and antisense strand of thedsRNA agent may otherwise be completely modified. The introduction ofthese motifs interrupts the modification pattern, if present, of thesense and/or antisense strand. The dsRNA agent optionally conjugateswith a GalNAc derivative ligand, for instance on the sense strand. Theresulting dsRNA agents present superior gene silencing activity.

The inventors surprisingly discovered that having one or more motifs ofthree identical modifications on three consecutive nucleotides at ornear the cleavage site of at least one strand of a dsRNA agentsuperiorly enhanced the gene silencing activity of the dsRNA agent.

Accordingly, the invention provides a double-stranded RNAi (dsRNA) agentcapable of inhibiting the expression of a target gene. The dsRNA agentcomprises a sense strand and an antisense strand. Each strand of thedsRNA agent can range from 12-30 nucleotides in length. For example,each strand can be between 14-30 nucleotides in length, 17-30nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides inlength, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides inlength, 19-21 nucleotides in length, 21-25 nucleotides in length, or21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex dsRNA. Theduplex region of a dsRNA agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 25-30 nucleotides in length,27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length,17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length,19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length,19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or21-23 nucleotide pairs in length. In another example, the duplex regionis selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27.

In one embodiment, the dsRNA agent of the invention comprises maycontain one or more overhang regions and/or capping groups of dsRNAagent at the 3′-end, or 5′-end or both ends of a strand. The overhangcan be 1-6 nucleotides in length, for instance 2-6 nucleotides inlength, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides inlength, 2-3 nucleotides in length, or 1-2 nucleotides in length. Theoverhangs can be the result of one strand being longer than the other,or the result of two strands of the same length being staggered. Theoverhang can form a mismatch with the target mRNA or it can becomplementary to the gene sequences being targeted or can be othersequence. The first and second strands can also be joined, e.g., byadditional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the dsRNAagent of the invention can each independently be a modified orunmodified nucleotide including, but no limited to 2′-sugar modified,such as, 2-F 2′-Omethyl, thymidine (T),2′-β-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine(Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinationsthereof. For example, TT can be an overhang sequence for either end oneither strand. The overhang can form a mismatch with the target mRNA orit can be complementary to the gene sequences being targeted or can beother sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the dsRNA agent of the invention may be phosphorylated. Insome embodiments, the overhang region contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The dsRNA agent of the invention comprises only single overhang, whichcan strengthen the interference activity of the dsRNA, without affectingits overall stability. For example, the single-stranded overhang islocated at the 3′-terminal end of the sense strand or, alternatively, atthe 3′-terminal end of the antisense strand. The dsRNA may also have ablunt end, located at the 5′-end of the antisense strand (or the 3′-endof the sense strand) or vice versa. Generally, the antisense strand ofthe dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. While not bound by theory, the asymmetric blunt end at the 5′-endof the antisense strand and 3′-end overhang of the antisense strandfavor the guide strand loading into RISC process.

In one embodiment, the dsRNA agent of the invention may also have twoblunt ends, at both ends of the dsRNA duplex.

In one embodiment, the dsRNA agent of the invention is a double endedbluntmer of 19 nt in length, wherein the sense strand contains at leastone motif of three 2′-F modifications on three consecutive nucleotidesat positions 7,8,9 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11,12,13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention is a double endedbluntmer of 20 nt in length, wherein the sense strand contains at leastone motif of three 2′-F modifications on three consecutive nucleotidesat positions 8,9,10 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11,12,13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention is a double endedbluntmer of 21 nt in length, wherein the sense strand contains at leastone motif of three 2′-F modifications on three consecutive nucleotidesat positions 9,10,11 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11,12,13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention comprises a 21nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense,wherein the sense strand contains at least one motif of three 2′-Fmodifications on three consecutive nucleotides at positions 9,10,11 fromthe 5′ end; the antisense strand contains at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at positions11,12,13 from the 5′ end, wherein one end of the dsRNA is blunt, whilethe other end is comprises a 2 nt overhang. Preferably, the 2 ntoverhang is at the 3′-end of the antisense. Optionally, the dsRNAfurther comprises a ligand (preferably GalNAc₃).

In one embodiment, the dsRNA agent of the invention comprising a senseand antisense strands, wherein: the sense strand is 25-30 nucleotideresidues in length, wherein starting from the 5′ terminal nucleotide(position 1) positions 1 to 23 of said first strand comprise at least 8ribonucleotides; antisense strand is 36-66 nucleotide residues in lengthand, starting from the 3′ terminal nucleotide, comprises at least 8ribonucleotides in the positions paired with positions 1-23 of sensestrand to form a duplex; wherein at least the 3′ terminal nucleotide ofantisense strand is unpaired with sense strand, and up to 6 consecutive3′ terminal nucleotides are unpaired with sense strand, thereby forminga 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′terminus of antisense strand comprises from 10-30 consecutivenucleotides which are unpaired with sense strand, thereby forming a10-30 nucleotide single stranded 5′ overhang; wherein at least the sensestrand 5′ terminal and 3′ terminal nucleotides are base paired withnucleotides of antisense strand when sense and antisense strands arealigned for maximum complementarity, thereby forming a substantiallyduplexed region between sense and antisense strands; and antisensestrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of antisense strand length to reduce target geneexpression when said double stranded nucleic acid is introduced into amammalian cell; and wherein the sense strand contains at least one motifof three 2′-F modifications on three consecutive nucleotides, where atleast one of the motifs occurs at or near the cleavage site. Theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at or near the cleavagesite.

In one embodiment, the dsRNA agent of the invention comprising a senseand antisense strands, wherein said dsRNA agent comprises a first strandhaving a length which is at least 25 and at most 29 nucleotides and asecond strand having a length which is at most 30 nucleotides with atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at position 11,12,13 from the 5′ end; wherein said 3′ end ofsaid first strand and said 5′ end of said second strand form a blunt endand said second strand is 1-4 nucleotides longer at its 3′ end than thefirst strand, wherein the duplex region which is at least 25 nucleotidesin length, and said second strand is sufficiently complementary to atarget mRNA along at least 19 nt of said second strand length to reducetarget gene expression when said dsRNA agent is introduced into amammalian cell, and wherein dicer cleavage of said dsRNA preferentiallyresults in an siRNA comprising said 3′ end of said second strand,thereby reducing expression of the target gene in the mammal.Optionally, the dsRNA agent further comprises a ligand.

In one embodiment, the sense strand of the dsRNA agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the dsRNA agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand.

For dsRNA agent having a duplex region of 17-23 nt in length, thecleavage site of the antisense strand is typically around the 10, 11 and12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe dsRNA from the 5′-end.

The sense strand of the dsRNA agent comprises at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesof the motifs from both strands may overlap, or all three nucleotidesmay overlap.

In one embodiment, the sense strand of the dsRNA agent comprises morethan one motif of three identical modifications on three consecutivenucleotides. The first motif should occur at or near the cleavage siteof the strand and the other motifs may be a wing modifications. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradjacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thechemistry of the motifs are distinct from each other and when the motifsare separated by one or more nucleotide the chemistries of the motifscan be the same or different. Two or more wing modifications may bepresent. For instance, when two wing modifications are present, the wingmodifications may both occur at one end of the duplex region relative tothe first motif which is at or near the cleavage site or each of thewing modifications may occur on either side of the first motif.

Like the sense strand, the antisense strand of the dsRNA agent comprisesat least two motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that is present on the sense strand.

In one embodiment, the wing modification on the sense strand, antisensestrand, or both strands of the dsRNA agent typically does not includethe first one or two terminal nucleotides at the 3′-end, 5′-end or bothends of the strand.

In another embodiment, the wing modification on the sense strand,antisense strand, or both strands of the dsRNA agent typically does notinclude the first one or two paired nucleotides within the duplex regionat the 3′-end, 5′-end or both ends of the strand.

When the sense strand and the antisense strand of the dsRNA agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the dsRNA agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be aligned so that two wing modifications each fromone strand fall on one end of the duplex region, having an overlap ofone, two or three nucleotides; two modifications each from one strandfall on the other end of the duplex region, having an overlap of one,two or three nucleotides.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the dsRNA agent, including the nucleotides that are part ofthe motifs, may be modified. Each nucleotide may be modified with thesame or different modification which can include one or more alterationof one or both of the non-linking phosphate oxygens and/or of one ormore of the linking phosphate oxygens; alteration of a constituent ofthe ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar;wholesale replacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking 0 of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. E.g., a phosphorothioate modification at a non-linking 0 positionmay only occur at one or both termini, may only occur in a terminalregion, e.g., at a position on a terminal nucleotide or in the last 2,3, 4, 5, or 10 nucleotides of a strand, or may occur in double strandand single strand regions, particularly at termini. The 5′ end or endscan be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. E.g., it can be desirable to include purine nucleotides inoverhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-β-allyl, 2′-C— allyl, 2′-deoxy, or 2′-fluoro. Thestrands can contain more than one modification. In one embodiment, eachresidue of the sense strand and antisense strand is independentlymodified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the sense strand and antisense strand each containstwo differently modified nucleotides selected from 2′-O-methyl or2′-fluoro.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with 2′-O-methyl nucleotide, 2′-deoxyfluoronucleotide, 2′-O—N-methylacetamido (2′-O-NMA) nucleotide, a2′-O-dimethylaminoethoxyethyl (2′-β-DMAEOE) nucleotide, 2′-O-aminopropyl(2′-O-AP) nucleotide, or 2′-ara-F nucleotide.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” or “alternativepattern” as used herein refers to a motif having one or moremodifications, each modification occurring on alternating nucleotides ofone strand. The alternating nucleotide may refer to one per every othernucleotide or one per every three nucleotides, or a similar pattern. Forexample, if A, B and C each represent one type of modification to thenucleotide, the alternating motif can be “ABABABABABAB . . . ,”“AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,”“AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications of analternating pattern. The term “alternating motif” or “alternativepattern” as used herein refers to a motif having one or moremodifications, each modification occurring on alternating nucleotides ofone strand. The alternating nucleotide may refer to one per every othernucleotide or one per every three nucleotides, or a similar pattern. Forexample, if A, B and C each represent one type of modification to thenucleotide, the alternating motif can be “ABABABABABAB . . . ,”“AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,”“AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the dsRNA agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 3′-5′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisenese strand may start with “BBAABBAA”from 3′-5′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the dsRNA agent of the invention comprises thepattern of the alternating motif of 2′-O-methyl modification and 2′-Fmodification on the sense strand initially has a shift relative to thepattern of the alternating motif of 2′-O-methyl modification and 2′-Fmodification on the antisense strand initially, i.e., the 2′-O-methylmodified nucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The dsRNA agent of the invention may further comprise at least onephosphorothioate or methylphosphonate internucleotide linkage. Thephosphorothioate or methylphosphonate internucleotide linkagemodification may occur on any nucleotide of the sense strand orantisense strand or both in any position of the strand. For instance,the internucleotide linkage modification may occur on every nucleotideon the sense strand and/or antisense strand; each internucleotidelinkage modification may occur in an alternating pattern on the sensestrand or antisense strand; or the sense strand or antisense strandcomprises both internucleotide linkage modifications in an alternatingpattern. The alternating pattern of the internucleotide linkagemodification on the sense strand may be the same or different from theantisense strand, and the alternating pattern of the internucleotidelinkage modification on the sense strand may have a shift relative tothe alternating pattern of the internucleotide linkage modification onthe antisense strand.

In one embodiment, the dsRNA comprises the phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region comprises two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within duplex region. For example, at least 2, 3, 4, or allthe overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. Preferably, these terminal three nucleotides may be at the3′-end of the antisense strand.

In one embodiment the sense strand of the dsRNA comprises 1-10 blocks oftwo to ten phosphorothioate or methylphosphonate internucleotidelinkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or 16 phosphate internucleotide linkages, wherein one of thephosphorothioate or methylphosphonate internucleotide linkages is placedat any position in the oligonucleotide sequence and the said sensestrand is paired with an antisense strand comprising any combination ofphosphorothioate, methylphosphonate and phosphate internucleotidelinkages or an antisense strand comprising either phosphorothioate ormethylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocksof two phosphorothioate or methylphosphonate internucleotide linkagesseparated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,or 18 phosphate internucleotide linkages, wherein one of thephosphorothioate or methylphosphonate internucleotide linkages is placedat any position in the oligonucleotide sequence and the said antisensestrand is paired with a sense strand comprising any combination ofphosphorothioate, methylphosphonate and phosphate internucleotidelinkages or an antisense strand comprising either phosphorothioate ormethylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocksof three phosphorothioate or methylphosphonate internucleotide linkagesseparated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16phosphate internucleotide linkages, wherein one of the phosphorothioateor methylphosphonate internucleotide linkages is placed at any positionin the oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocksof four phosphorothioate or methylphosphonate internucleotide linkagesseparated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphateinternucleotide linkages, wherein one of the phosphorothioate ormethylphosphonate internucleotide linkages is placed at any position inthe oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocksof five phosphorothioate or methylphosphonate internucleotide linkagesseparated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphateinternucleotide linkages, wherein one of the phosphorothioate ormethylphosphonate internucleotide linkages is placed at any position inthe oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocksof six phosphorothioate or methylphosphonate internucleotide linkagesseparated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate internucleotidelinkages, wherein one of the phosphorothioate or methylphosphonateinternucleotide linkages is placed at any position in theoligonucleotide sequence and the said antisense strand is paired with asense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocksof seven phosphorothioate or methylphosphonate internucleotide linkagesseparated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphate internucleotidelinkages, wherein one of the phosphorothioate or methylphosphonateinternucleotide linkages is placed at any position in theoligonucleotide sequence and the said antisense strand is paired with asense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocksof eight phosphorothioate or methylphosphonate internucleotide linkagesseparated by 1, 2, 3, 4, 5 or 6 phosphate internucleotide linkages,wherein one of the phosphorothioate or methylphosphonate internucleotidelinkages is placed at any position in the oligonucleotide sequence andthe said antisense strand is paired with a sense strand comprising anycombination of phosphorothioate, methylphosphonate and phosphateinternucleotide linkages or an antisense strand comprising eitherphosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocksof nine phosphorothioate or methylphosphonate internucleotide linkagesseparated by 1, 2, 3 or 4 phosphate internucleotide linkages, whereinone of the phosphorothioate or methylphosphonate internucleotidelinkages is placed at any position in the oligonucleotide sequence andthe said antisense strand is paired with a sense strand comprising anycombination of phosphorothioate, methylphosphonate and phosphateinternucleotide linkages or an antisense strand comprising eitherphosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment, the dsRNA of the invention further comprises one ormore phosphorothioate or methylphosphonate internucleotide linkagemodification within 1-10 of the termini position(s) of the sense and/orantisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10nucleotides may be linked through phosphorothioate or methylphosphonateinternucleotide linkage at one end or both ends of the sense and/orantisense strand.

In one embodiment, the dsRNA of the invention further comprises one ormore phosphorothioate or methylphosphonate internucleotide linkagemodification within 1-10 of the internal region of the duplex of each ofthe sense and/or antisense strand. For example, at least 2, 3, 4, 5, 6,7, 8, 9 or 10 nucleotides may be linked through phosphorothioatemethylphosphonate internucleotide linkage at position 8-16 of the duplexregion counting from the 5′-end of the sense strand; the dsRNA canoptionally further comprise one or more phosphorothioate ormethylphosphonate internucleotide linkage modification within 1-10 ofthe termini position(s).

In one embodiment, the dsRNA of the invention further comprises one tofive phosphorothioate or methylphosphonate internucleotide linkagemodification(s) within position 1-5 and one to five phosphorothioate ormethylphosphonate internucleotide linkage modification(s) withinposition 18-23 of the sense strand (counting from the 5′-end), and oneto five phosphorothioate or methylphosphonate internucleotide linkagemodification at positions 1 and 2 and one to five within positions 18-23of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises onephosphorothioate internucleotide linkage modification within position1-5 and one phosphorothioate or methylphosphonate internucleotidelinkage modification within position 18-23 of the sense strand (countingfrom the 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and 2 and two phosphorothioate ormethylphosphonate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications within position1-5 and one phosphorothioate internucleotide linkage modification withinposition 18-23 of the sense strand (counting from the 5′-end), and onephosphorothioate internucleotide linkage modification at positions 1 and2 and two phosphorothioate internucleotide linkage modifications withinpositions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications within position1-5 and two phosphorothioate internucleotide linkage modificationswithin position 18-23 of the sense strand (counting from the 5′-end),and one phosphorothioate internucleotide linkage modification atpositions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications within position1-5 and two phosphorothioate internucleotide linkage modificationswithin position 18-23 of the sense strand (counting from the 5′-end),and one phosphorothioate internucleotide linkage modification atpositions 1 and 2 and one phosphorothioate internucleotide linkagemodification within positions 18-23 of the antisense strand (countingfrom the 5′-end).

In one embodiment, the dsRNA of the invention further comprises onephosphorothioate internucleotide linkage modification within position1-5 and one phosphorothioate internucleotide linkage modification withinposition 18-23 of the sense strand (counting from the 5′-end), and twophosphorothioate internucleotide linkage modifications at positions 1and 2 and two phosphorothioate internucleotide linkage modificationswithin positions 18-23 of the antisense strand (counting from the5′-end).

In one embodiment, the dsRNA of the invention further comprises onephosphorothioate internucleotide linkage modification within position1-5 and one within position 18-23 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationat positions 1 and 2 and one phosphorothioate internucleotide linkagemodification within positions 18-23 of the antisense strand (countingfrom the 5′-end).

In one embodiment, the dsRNA of the invention further comprises onephosphorothioate internucleotide linkage modification within position1-5 (counting from the 5′-end), and two phosphorothioateinternucleotidelinkage modifications at positions 1 and 2 and one phosphorothioateinternucleotide linkage modification within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications within position1-5 (counting from the 5′-end), and one phosphorothioateinternucleotidelinkage modification at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications within position1-5 and one within position 18-23 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and one phosphorothioate internucleotide linkagemodification within positions 18-23 of the antisense strand (countingfrom the 5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications within position1-5 and one phosphorothioate internucleotide linkage modification withinposition 18-23 of the sense strand (counting from the 5′-end), and twophosphorothioate internucleotide linkage modifications at positions 1and 2 and two phosphorothioate internucleotide linkage modificationswithin positions 18-23 of the antisense strand (counting from the5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications within position1-5 and one phosphorothioate internucleotide linkage modification withinposition 18-23 of the sense strand (counting from the 5′-end), and onephosphorothioate internucleotide linkage modification at positions 1 and2 and two phosphorothioate internucleotide linkage modifications withinpositions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications at position 1 and2, and two phosphorothioate internucleotide linkage modifications atposition 20 and 21 of the sense strand (counting from the 5′-end), andone phosphorothioate internucleotide linkage modification at positions 1and one at position 21 of the antisense strand (counting from the5′-end).

In one embodiment, the dsRNA of the invention further comprises onephosphorothioate internucleotide linkage modification at position 1, andone phosphorothioate internucleotide linkage modification at position 21of the sense strand (counting from the 5′-end), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications at positions 20and 21 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications at position 1 and2, and two phosphorothioate internucleotide linkage modifications atposition 21 and 22 of the sense strand (counting from the 5′-end), andone phosphorothioate internucleotide linkage modification at positions 1and one phosphorothioate internucleotide linkage modification atposition 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises onephosphorothioate internucleotide linkage modification at position 1, andone phosphorothioate internucleotide linkage modification at position 21of the sense strand (counting from the 5′-end), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications at positions 21and 22 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises twophosphorothioate internucleotide linkage modifications at position 1 and2, and two phosphorothioate internucleotide linkage modifications atposition 22 and 23 of the sense strand (counting from the 5′-end), andone phosphorothioate internucleotide linkage modification at positions 1and one phosphorothioate internucleotide linkage modification atposition 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises onephosphorothioate internucleotide linkage modification at position 1, andone phosphorothioate internucleotide linkage modification at position 21of the sense strand (counting from the 5′-end), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications at positions 23and 23 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention comprisesmismatch(es) with the target, within the duplex, or combinationsthereof. The mistmatch can occur in the overhang region or the duplexregion. The base pair can be ranked on the basis of their propensity topromote dissociation or melting (e.g., on the free energy of associationor dissociation of a particular pairing, the simplest approach is toexamine the pairs on an individual pair basis, though next neighbor orsimilar analysis can also be used). In terms of promoting dissociation:A:U is preferred over G:C; G:U is preferred over G:C; and I:C ispreferred over G:C (I=inosine). Mismatches, e.g., non-canonical or otherthan canonical pairings (as described elsewhere herein) are preferredover canonical (A:T, A:U, G:C) pairings; and pairings which include auniversal base are preferred over canonical pairings.

In one embodiment, the dsRNA agent of the invention comprises at leastone of the first 1, 2, 3, 4, or 5 base pairs within the duplex regionsfrom the 5′-end of the antisense strand can be chosen independently fromthe group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonicalor other than canonical pairings or pairings which include a universalbase, to promote the dissociation of the antisense strand at the 5′-endof the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In one embodiment, the sense strand sequence may be represented byformula (I):

(I) 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein N_(b) and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the dsRNA agent has a duplex regionof 17-23 nucleotide pairs in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

(Ia) 5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′; (Ib) 5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′; or (Ic) 5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ia), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In one embodiment, the antisense strand sequence of the dsRNA may berepresented by formula (II):

(II) 5′ n_(q)′-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)₁-N′_(a)-n_(p)′ 3′wherein:

k and l are each independently 0 or 1;

p and q are each independently 0-6; each N_(a)′ independently representsan oligonucleotide sequence comprising 0-25 modified nucleotides, eachsequence comprising at least two differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotidecomprising 0-6 nucleotides;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the dsRNA agent has a duplex region of 17-23nt in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11,12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, withthe count starting from the 1^(st) nucleotide, from the 5′-end; oroptionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occursat positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:

(IIa) 5′ n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p)′ 3′; (IIb) 5′n_(q)′-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p)′ 3′; or (IIc) 5′n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p)′ 3′.

When the antisense strand is represented by formula (IIa), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C— allyl, or 2′-fluoro. For example, eachnucleotide of the sense strand and antisense strand is independentlymodified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, inparticular, may represent a 2′-O-methyl modification or a 2′-fluoromodification.

In one embodiment, the sense strand of the dsRNA agent comprises YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia), (Ib)and (Ic) forms a duplex with a antisense strand being represented by anyone of formulas (IIa), (IIb) and (IIc), respectively.

Accordingly, the dsRNA agent may comprise a sense strand and anantisense strand, each strand having 14 to 30 nucleotides, the dsRNAduplex represented by formula (III):

(III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)₁-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q) independently represents anoverhang nucleotide sequence; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both iand j are 1. In another embodiment, k is 1 and l is 0; k is 0 and l is1; or both k and l are 1.

In one embodiment, the dsRNA agent of the invention comprises a sensestrand and an antisense strand, each strand having 14 to 30 nucleotides,the dsRNA duplex represented by formula (V):

(V) sense: 5′N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)₁-N_(a)′ 5′

wherein:

j, k, and 1 are each independently 0 or 1;

p and q are each independently 2;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, and n_(q) independently represents an overhang nucleotidesequence; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both iand j are 1. In another embodiment, k is 1 and l is 0; k is 0 and l is1; or both k and l are 1.

In one embodiment, the dsRNA agent of the invention comprises a sensestrand and an antisense strand, each strand having 14 to 30 nucleotides,the dsRNA duplex represented by formula (Va):

(Va) sense: 5′ N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a) 3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)₁-N_(a)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p and q are each independently 2;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

n_(p)′ represents an overhang nucleotide sequence; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

Exemplary combinations of the sense strand and antisense strand forminga dsRNA duplex include the formulas below:

(IIIa) 5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ 3′n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′n_(q)′ 5′ (IIIb) 5′n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′ 3′n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIIc) 5′n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-nq 3′ 3′n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n_(q)′ 5′

When the dsRNA agent is represented by formula (IIIa), each N_(b) andN_(b)′ independently represents an oligonucleotide sequence comprising1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) and N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the dsRNA agent is represented as formula (IIIb), each N_(b) andN_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. EachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNA agent is represented as formula (IIIc), each N_(b) andN_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. EachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a),N_(a)′, N_(b) and N_(b)′ independently comprises modifications ofalternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb) and (IIc) may bethe same or different from each other.

When the dsRNA agent is represented by formula (III), (IIIa), (IIIb) or(IIc), at least one of the Y nucleotides may form a base pair with oneof the Y′ nucleotides. Alternatively, at least two of the Y nucleotidesform base pairs with the corresponding Y′ nucleotides; or all three ofthe Y nucleotides all form base pairs with the corresponding Y′nucleotides.

It is understood that N_(a) nucleotides from base pair with N_(a)′,N_(b) nucleotides from base pair with N_(b)′, X nucleotides from basepair with X′, Y nucleotides from base pair with Y′, and Z nucleotidesfrom base pair with Z′.

When the dsRNA agent is represented by formula (IIIa) or (IIIc), atleast one of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNA agent is represented as formula (IIIb) or (IIIc), atleast one of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, the dsRNA agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb) or (IIIc), whereinsaid duplexes are connected by a linker. The linker can be cleavable ornon-cleavable. Optionally, said multimer further comprise a ligand. Eachof the dsRNA can target the same gene or two different genes; or each ofthe dsRNA can target same gene at two different target sites.

In one embodiment, the dsRNA agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb)or (IIIc), wherein said duplexes are connected by a linker. The linkercan be cleavable or non-cleavable. Optionally, said multimer furthercomprises a ligand. Each of the dsRNA can target the same gene or twodifferent genes; or each of the dsRNA can target same gene at twodifferent target sites.

In one embodiment, two dsRNA agent represented by formula (III), (IIIa),(IIIb) or (IIIc) are linked to each other at the 5′ end, and one or bothof the 3′ ends of the are optionally conjugated to a ligand. Each of thedsRNA can target the same gene or two different genes; or each of thedsRNA can target same gene at two different target sites.

Various publications described multimeric siRNA and can all be used withthe dsRNA of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 which are hereby incorporated by their entirely.

The dsRNA agent that contains conjugations of one or more carbohydratemoieties to a dsRNA agent can optimize one or more properties of thedsRNA agent. In many cases, the carbohydrate moiety will be attached toa modified subunit of the dsRNA agent. E.g., the ribose sugar of one ormore ribonucleotide subunits of a dsRNA agent can be replaced withanother moiety, e.g., a non-carbohydrate (preferably cyclic) carrier towhich is attached a carbohydrate ligand. A ribonucleotide subunit inwhich the ribose sugar of the subunit has been so replaced is referredto herein as a ribose replacement modification subunit (RRMS). A cycliccarrier may be a carbocyclic ring system, i.e., all ring atoms arecarbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

In embodiment the dsRNA of the invention is conjugated to a ligand via acarrier, wherein the carrier can be cyclic group or acyclic group;preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,[1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and decalin; preferably, the acyclic group is selectedfrom serinol backbone or diethanolamine backbone.

The double-stranded RNA (dsRNA) agent of the invention may optionally beconjugated to one or more ligands. The ligand can be attached to thesense strand, antisense strand or both strands, at the 3′-end, 5′-end orboth ends. For instance, the ligand may be conjugated to the sensestrand, in particular, the 3′-end of the sense strand.

Ligands

A wide variety of entities can be coupled to the oligonucleotides of thepresent invention. Preferred moieties are ligands, which are coupled,preferably covalently, either directly or indirectly via an interveningtether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, receptor e.g., acellular or organ compartment, tissue, organ or region of the body, as,e.g., compared to a species absent such a ligand. Ligands providingenhanced affinity for a selected target are also termed targetingligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In one embodiment, the endosomolytic ligand assumes itsactive conformation at endosomal pH. The “active” conformation is thatconformation in which the endosomolytic ligand promotes lysis of theendosome and/or transport of the composition of the invention, or itscomponents, from the endosome to the cytoplasm of the cell. Exemplaryendosomolytic ligands include the GALA peptide (Subbarao et al.,Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J.Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, theendosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched.

Ligands can improve transport, hybridization, and specificity propertiesand may also improve nuclease resistance of the resultant natural ormodified oligoribonucleotide, or a polymeric molecule comprising anycombination of monomers described herein and/or natural or modifiedribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 2 shows some examples of targetingligands and their associated receptors.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases or a chelator(e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O (hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the oligonucleotide into the cellby activating an inflammatory response, for example. Exemplary ligandsthat would have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, naproxen or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alphα-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. In another alternative, the peptide moiety caninclude a hydrophobic membrane translocation sequence (MTS). Anexemplary hydrophobic MTS-containing peptide is RFGF having the aminoacid sequence AAVALLPAVLLALLAP (SEQ ID NO: 1). An RFGF analogue (e.g.,amino acid sequence AALLPVLLAAP (SEQ ID NO: 2)) containing a hydrophobicMTS can also be a targeting moiety. The peptide moiety can be a“delivery” peptide, which can carry large polar molecules includingpeptides, oligonucleotides, and protein across cell membranes. Forexample, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) (SEQ ID NO:3) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ IDNO: 4) have been found to be capable of functioning as deliverypeptides. A peptide or peptidomimetic can be encoded by a randomsequence of DNA, such as a peptide identified from a phage-displaylibrary, or one-bead-one-compound (OBOC) combinatorial library (Lam etal., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetictethered to an iRNA agent via an incorporated monomer unit is a celltargeting peptide such as an arginine-glycine-aspartic acid(RGD)-peptide, or RGD mimic. A peptide moiety can range in length fromabout 5 amino acids to about 40 amino acids. The peptide moieties canhave a structural modification, such as to increase stability or directconformational properties. Any of the structural modifications describedbelow can be utilized. An RGD peptide moiety can be used to target atumor cell, such as an endothelial tumor cell or a breast cancer tumorcell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptidecan facilitate targeting of an iRNA agent to tumors of a variety ofother tissues, including the lung, kidney, spleen, or liver (Aoki etal., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptidewill facilitate targeting of an iRNA agent to the kidney. The RGDpeptide can be linear or cyclic, and can be modified, e.g., glycosylatedor methylated to facilitate targeting to specific tissues. For example,a glycosylated RGD peptide can deliver an iRNA agent to a tumor cellexpressing α_(V)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an integrin. Thus, onecould use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the integrin ligand.Generally, such ligands can be used to control proliferating cells andangiogeneis. Preferred conjugates of this type lignads that targetsPECAM-1, VEGF, or other cancer gene, e.g., a cancer gene describedherein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide can be an amphipathic α-helicalpeptide. Exemplary amphipathic α-helical peptides include, but are notlimited to, cecropins, lycotoxins, paradaxins, buforin, CPF,bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clavapeptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂Apeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an apatamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycarboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Exemplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbaone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligand conjugates amenable to the invention are described in U.S.patent applications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S.Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934,filed Aug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 andU.S. Ser. No. 11/944,227 filed Nov. 21, 2007, which are incorporated byreference in their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides at various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether, e.g. a carrier described herein. The ligand ortethered ligand may be present on a monomer when said monomer isincorporated into the growing strand. In some embodiments, the ligandmay be incorporated via coupling to a “precursor” monomer after said“precursor” monomer has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether (i.e.,having no associated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporatedinto a growing oligonucleotide strand. In a subsequent operation, i.e.,after incorporation of the precursor monomer into the strand, a ligandhaving an electrophilic group, e.g., a pentafluorophenyl ester oraldehyde group, can subsequently be attached to the precursor monomer bycoupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor monomer's tether.

In another example, a monomer having a chemical group suitable fortaking part in Click Chemistry reaction may be incorporated e.g., anazide or alkyne terminated tether/linker. In a subsequent operation,i.e., after incorporation of the precursor monomer into the strand, aligand having complementary chemical group, e.g. an alkyne or azide canbe attached to the precursor monomer by coupling the alkyne and theazide together.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

Any suitable ligand in the field of RNA interference may be used,although the ligand is typically a carbohydrate e.g. monosaccharide(such as GalNAc), disaccharide, trisaccharide, tetrasaccharide,polysaccharide.

Linkers that conjugate the ligand to the nucleic acid include thosediscussed above. For example, the ligand can be one or more GalNAc(N-acetylglucosamine) derivatives attached through a bivalent ortrivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to abivalent and trivalent branched linkers include the structures shown inany of formula (IV)-(VII):

wherein:

q^(2A), q^(2B), q^(3A), q^(3B), q^(4A), q^(4B), q^(5B) and q^(5C) foreach represent independently occurrence 0-20 and wherein the repeatingunit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), CC or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and

R^(a) is H or amino acid side chain.

Trivalent conjugating GalNAc derivatives are particularly useful for usewith RNAi agents for inhibiting the expression of a target gene, such asthose of formula (VII):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thefollowing compounds:

Definitions

As used herein, the terms “dsRNA”, “siRNA”, and “iRNA agent” are usedinterchangeably to agents that can mediate silencing of a target RNA,e.g., mRNA, e.g., a transcript of a gene that encodes a protein. Forconvenience, such mRNA is also referred to herein as mRNA to besilenced. Such a gene is also referred to as a target gene. In general,the RNA to be silenced is an endogenous gene or a pathogen gene. Inaddition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also betargeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an siRNA agent of 21 to 23nucleotides.

As used herein, “specifically hybridizable” and “complementary” areterms which are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between a compound of theinvention and a target RNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of assays or therapeutic treatment, or in the case of invitro assays, under conditions in which the assays are performed. Thenon-target sequences typically differ by at least 5 nucleotides.

In one embodiment, a dsRNA agent of the invention is “sufficientlycomplementary” to a target RNA, e.g., a target mRNA, such that the dsRNAagent silences production of protein encoded by the target mRNA. Inanother embodiment, the dsRNA agent of the invention is “exactlycomplementary” to a target RNA, e.g., the target RNA and the dsRNAduplex agent anneal, for example to form a hybrid made exclusively ofWatson-Crick base pairs in the region of exact complementarity. A“sufficiently complementary” target RNA can include an internal region(e.g., of at least 10 nucleotides) that is exactly complementary to atarget RNA. Moreover, in some embodiments, the dsRNA agent of theinvention specifically discriminates a single-nucleotide difference. Inthis case, the dsRNA agent only mediates RNAi if exact complementary isfound in the region (e.g., within 7 nucleotides of) thesingle-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acidmolecule (RNA or DNA) for example of length less than 100, 200, 300, or400 nucleotides.

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine. The term “alkyl” refers to saturated and unsaturatednon-aromatic hydrocarbon chains that may be a straight chain or branchedchain, containing the indicated number of carbon atoms (these includewithout limitation propyl, allyl, or propargyl), which may be optionallyinserted with N, O, or S. For example, C₁-C₁₀ indicates that the groupmay have from 1 to 10 (inclusive) carbon atoms in it. The term “alkoxy”refers to an —O-alkyl radical. The term “alkylene” refers to a divalentalkyl (i.e., —R—). The term “alkylenedioxo” refers to a divalent speciesof the structure —O—R—O—, in which R represents an alkylene. The term“aminoalkyl” refers to an alkyl substituted with an amino The term“mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an—S-alkyl radical.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include trizolyl, tetrazolyl, piperazinyl,pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl,arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl,alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylamino carbonyl,arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino,trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl,arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl,carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl,heteroaryl, heterocyclic, and aliphatic. It is understood that thesubstituent can be further substituted.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above. As used herein,“carbohydrate” refers to a compound which is either a carbohydrate perse made up of one or more monosaccharide units having at least 6 carbonatoms (which may be linear, branched or cyclic) with an oxygen, nitrogenor sulfur atom bonded to each carbon atom; or a compound having as apart thereof a carbohydrate moiety made up of one or more monosaccharideunits each having at least six carbon atoms (which may be linear,branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded toeach carbon atom. Representative carbohydrates include the sugars(mono-, di-, tri- and oligosaccharides containing from about 4-9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C₅and above (preferably C₅-C₈) sugars; di- and trisaccharides includesugars having two or three monosaccharide units (preferably C₅-C₈).

Alternative Embodiments

In another embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 30nucleotides. The sense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site in the antisensestrand. Every nucleotide in the sense strand and antisense strand hasbeen modified. The modifications on sense strand and antisense strandeach independently comprises at least two different modifications.

In another embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 30nucleotides. The sense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site in the antisensestrand. The antisense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides. Themodification pattern of the antisense strand is shifted by one or morenucleotides relative to the modification pattern of the sense strand.

In another embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 30nucleotides. The sense strand contains at least two motifs of threeidentical modifications on three consecutive nucleotides, when at leastone of the motifs occurs at the cleavage site in the strand and at leastone of the motifs occurs at another portion of the strand that isseparated from the motif at the cleavage site by at least onenucleotide. The antisense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site in the strand andat least one of the motifs occurs at another portion of the strand thatis separated from the motif at or near cleavage site by at least onenucleotide.

In another embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 30nucleotides. The sense strand contains at least two motifs of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at the cleavage site in the strand and at leastone of the motifs occurs at another portion of the strand that isseparated from the motif at the cleavage site by at least onenucleotide. The antisense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site in the strand andat least one of the motifs occurs at another portion of the strand thatis separated from the motif at or near cleavage site by at least onenucleotide. The modification in the motif occurring at the cleavage sitein the sense strand is different than the modification in the motifoccurring at or near the cleavage site in the antisense strand. Inanother embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 12 to 30nucleotides. The sense strand contains at least one motif of three 2′-Fmodifications on three consecutive nucleotides, where at least one ofthe motifs occurs at the cleavage site in the strand. The antisensestrand contains at least one motif of three 2′-O-methyl modifications onthree consecutive nucleotides.

The sense strand may further comprises one or more motifs of threeidentical modifications on three consecutive nucleotides, where the oneor more additional motifs occur at another portion of the strand that isseparated from the three 2′-F modifications at the cleavage site by atleast one nucleotide. The antisense strand may further comprises one ormore motifs of three identical modifications on three consecutivenucleotides, where the one or more additional motifs occur at anotherportion of the strand that is separated from the three 2′-O-methylmodifications by at least one nucleotide. At least one of thenucleotides having a 2′-F modification may form a base pair with one ofthe nucleotides having a 2′-O-methyl modification.

In one embodiment, the dsRNA of the invention is administered in buffer.

In one embodiment, siRNA compounds described herein can be formulatedfor administration to a subject. A formulated siRNA composition canassume a variety of states. In some examples, the composition is atleast partially crystalline, uniformly crystalline, and/or anhydrous(e.g., less than 80, 50, 30, 20, or 10% water). In another example, thesiRNA is in an aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the siRNAcomposition is formulated in a manner that is compatible with theintended method of administration, as described herein. For example, inparticular embodiments the composition is prepared by at least one ofthe following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A siRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a siRNA,e.g., a protein that complexes with siRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the siRNA preparation includes another siNA compound,e.g., a second siRNA that can mediate RNAi with respect to a secondgene, or with respect to the same gene. Still other preparation caninclude at least 3, 5, ten, twenty, fifty, or a hundred or moredifferent siRNA species. Such siRNAs can mediate RNAi with respect to asimilar number of different genes.

In one embodiment, the siRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than a RNA or a DNA). Forexample, a siRNA composition for the treatment of a viral disease, e.g.,HIV, might include a known antiviral agent (e.g., a protease inhibitoror reverse transcriptase inhibitor). In another example, a siRNAcomposition for the treatment of a cancer might further comprise achemotherapeutic agent.

Exemplary formulations are discussed below.

Liposomes.

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified siRNAcompounds. It may be understood, however, that these formulations,compositions and methods can be practiced with other siRNA compounds,e.g., modified siRNAs, and such practice is within the invention. AnsiRNA compound, e.g., a double-stranded siRNA compound, or ssiRNAcompound, (e.g., a precursor, e.g., a larger siRNA compound which can beprocessed into a ssiRNA compound, or a DNA which encodes an siRNAcompound, e.g., a double-stranded siRNA compound, or ssiRNA compound, orprecursor thereof) preparation can be formulated for delivery in amembranous molecular assembly, e.g., a liposome or a micelle. As usedherein, the term “liposome” refers to a vesicle composed of amphiphiliclipids arranged in at least one bilayer, e.g., one bilayer or aplurality of bilayers. Liposomes include unilamellar and multilamellarvesicles that have a membrane formed from a lipophilic material and anaqueous interior. The aqueous portion contains the siRNA composition.The lipophilic material isolates the aqueous interior from an aqueousexterior, which typically does not include the siRNA composition,although in some examples, it may. Liposomes are useful for the transferand delivery of active ingredients to the site of action. Because theliposomal membrane is structurally similar to biological membranes, whenliposomes are applied to a tissue, the liposomal bilayer fuses withbilayer of the cellular membranes. As the merging of the liposome andcell progresses, the internal aqueous contents that include the siRNAare delivered into the cell where the siRNA can specifically bind to atarget RNA and can mediate RNAi. In some cases the liposomes are alsospecifically targeted, e.g., to direct the siRNA to particular celltypes.

A liposome containing a siRNA can be prepared by a variety of methods.In one example, the lipid component of a liposome is dissolved in adetergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The siRNApreparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the siRNA andcondense around the siRNA to form a liposome. After condensation, thedetergent is removed, e.g., by dialysis, to yield a liposomalpreparation of siRNA.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Further description of methods for producing stable polynucleotidedelivery vehicles, which incorporate a polynucleotide/cationic lipidcomplex as structural components of the delivery vehicle, are describedin, e.g., WO 96/37194. Liposome formation can also include one or moreaspects of exemplary methods described in Felgner, P. L. et al., Proc.Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S.Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson,et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl.Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; andFukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques forpreparing lipid aggregates of appropriate size for use as deliveryvehicles include sonication and freeze-thaw plus extrusion (see, e.g.,Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidizationcan be used when consistently small (50 to 200 nm) and relativelyuniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta775:169, 1984). These methods are readily adapted to packaging siRNApreparations into liposomes.

Liposomes that are pH-sensitive or negatively-charged entrap nucleicacid molecules rather than complex with them. Since both the nucleicacid molecules and the lipid are similarly charged, repulsion ratherthan complex formation occurs. Nevertheless, some nucleic acid moleculesare entrapped within the aqueous interior of these liposomes.pH-sensitive liposomes have been used to deliver DNA encoding thethymidine kinase gene to cell monolayers in culture. Expression of theexogenous gene was detected in the target cells (Zhou et al., Journal ofControlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andinclude U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569;WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver siRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated siRNAs in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of siRNA (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Choi”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer siRNA, into the skin. In some implementations,liposomes are used for delivering siRNA to epidermal cells and also toenhance the penetration of siRNA into dermal tissues, e.g., into skin.For example, the liposomes can be applied topically. Topical delivery ofdrugs formulated as liposomes to the skin has been documented (see,e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino,R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T.et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176,1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527,1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith siRNA are useful for treating a dermatological disorder.

Liposomes that include siRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include siRNA can bedelivered, for example, subcutaneously by infection in order to deliversiRNA to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransferosomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. Nos. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Surfactants.

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified siRNAcompounds. It may be understood, however, that these formulations,compositions and methods can be practiced with other siRNA compounds,e.g., modified siRNA compounds, and such practice is within the scope ofthe invention. Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes (see above). siRNA(or a precursor, e.g., a larger dsiRNA which can be processed into asiRNA, or a DNA which encodes a siRNA or precursor) compositions caninclude a surfactant. In one embodiment, the siRNA is formulated as anemulsion that includes a surfactant. The most common way of classifyingand ranking the properties of the many different types of surfactants,both natural and synthetic, is by the use of the hydrophile/lipophilebalance (HLB). The nature of the hydrophilic group provides the mostuseful means for categorizing the different surfactants used informulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker,Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical products and are usable over a wide range of pH values.In general their HLB values range from 2 to about 18 depending on theirstructure. Nonionic surfactants include nonionic esters such as ethyleneglycol esters, propylene glycol esters, glyceryl esters, polyglycerylesters, sorbitan esters, sucrose esters, and ethoxylated esters.Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates,propoxylated alcohols, and ethoxylated/propoxylated block polymers arealso included in this class. The polyoxyethylene surfactants are themost popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and Other Membranous Formulations.

For ease of exposition the micelles and other formulations, compositionsand methods in this section are discussed largely with regard tounmodified siRNA compounds. It may be understood, however, that thesemicelles and other formulations, compositions and methods can bepracticed with other siRNA compounds, e.g., modified siRNA compounds,and such practice is within the invention. The siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor,e.g., a larger siRNA compound which can be processed into a ssiRNAcompound, or a DNA which encodes an siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, or precursorthereof)) composition can be provided as a micellar formulation.“Micelles” are defined herein as a particular type of molecular assemblyin which amphipathic molecules are arranged in a spherical structuresuch that all the hydrophobic portions of the molecules are directedinward, leaving the hydrophilic portions in contact with the surroundingaqueous phase. The converse arrangement exists if the environment ishydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxocholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, late, and mixtures thereof. The micelleforming compounds may be added at the same time or after addition of thealkali metal alkyl sulphate. Mixed micelles will form with substantiallyany kind of mixing of the ingredients but vigorous mixing in order toprovide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

Particles.

For ease of exposition the particles, formulations, compositions andmethods in this section are discussed largely with regard to modifiedsiRNA compounds. It may be understood, however, that these particles,formulations, compositions and methods can be practiced with other siRNAcompounds, e.g., unmodified siRNA compounds, and such practice is withinthe invention. In another embodiment, an siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor,e.g., a larger siRNA compound which can be processed into a ssiRNAcompound, or a DNA which encodes an siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, or precursorthereof) preparations may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying, but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.

Pharmaceutical Compositions

The iRNA agents of the invention may be formulated for pharmaceuticaluse. Pharmaceutically acceptable compositions comprise atherapeutically-effective amount of one or more of the dsRNA agents inany of the preceding embodiments, taken alone or formulated togetherwith one or more pharmaceutically acceptable carriers (additives),excipient and/or diluents.

The pharmaceutical compositions may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally. Delivery using subcutaneous or intravenous methods can beparticularly advantageous.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe invention which is effective for producing some desired therapeuticeffect in at least a sub-population of cells in an animal at areasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium state, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; and (22) other non-toxic compatible substancesemployed in pharmaceutical formulations.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 0.1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bio available a compound of the presentinvention.

iRNA agent preparation can be formulated in combination with anotheragent, e.g., another therapeutic agent or an agent that stabilizes aiRNA, e.g., a protein that complexes with iRNA to form an iRNP. Stillother agents include chelators, e.g., EDTA (e.g., to remove divalentcations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broadspecificity RNAse inhibitor such as RNAsin) and so forth.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure. The patient receiving this treatment is any animal in need,including primates, in particular humans, and other mammals such asequines, cattle, swine and sheep; and poultry and pets in general.

Double-stranded RNAi agents are produced in a cell in vivo, e.g., fromexogenous DNA templates that are delivered into the cell. For example,the DNA templates can be inserted into vectors and used as gene therapyvectors. Gene therapy vectors can be delivered to a subject by, forexample, intravenous injection, local administration (U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. The DNA templates, for example, caninclude two transcription units, one that produces a transcript thatincludes the top strand of a dsRNA agent and one that produces atranscript that includes the bottom strand of a dsRNA agent. When thetemplates are transcribed, the dsRNA agent is produced, and processedinto siRNA agent fragments that mediate gene silencing.

Routes of Delivery

A composition that includes an iRNA can be delivered to a subject by avariety of routes. Exemplary routes include: intravenous, subcutaneous,topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically include one or more species of iRNA and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The compositions of the present invention may be administered in anumber of ways depending upon whether local or systemic treatment isdesired and upon the area to be treated. Administration may be topical(including ophthalmic, vaginal, rectal, intranasal, transdermal), oralor parenteral. Parenteral administration includes intravenous drip,subcutaneous, intraperitoneal or intramuscular injection, or intrathecalor intraventricular administration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the iRNA in aerosol form. The vascularendothelial cells could be targeted by coating a balloon catheter withthe iRNA and mechanically introducing the DNA.

Dosage

In one aspect, the invention features a method of administering a dsRNAagent, e.g., a siRNA agent, to a subject (e.g., a human subject). Themethod includes administering a unit dose of the dsRNA agent, e.g., asiRNA agent, e.g., double stranded siRNA agent that (a) thedouble-stranded part is 14-30 nucleotides (nt) long, for example, 21-23nt, (b) is complementary to a target RNA (e.g., an endogenous orpathogen target RNA), and, optionally, (c) includes at least one 3′overhang 1-5 nucleotide long. In one embodiment, the unit dose is lessthan 10 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1,0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kgof bodyweight, and less than 200 nmole of RNA agent (e.g., about4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150,75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075,0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with thetarget RNA. The unit dose, for example, can be administered by injection(e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, ora topical application. In some embodiments dosages may be less than 10,5, 2, 1, or 0.1 mg/kg of body weight.

In some embodiments, the unit dose is administered less frequently thanonce a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities. In one embodiment, the subject has aviral infection and the modality is an antiviral agent other than adsRNA agent, e.g., other than a siRNA agent. In another embodiment, thesubject has atherosclerosis and the effective dose of a dsRNA agent,e.g., a siRNA agent, is administered in combination with, e.g., aftersurgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of a dsRNA agent, e.g., a siRNA agent, (e.g., aprecursor, e.g., a larger dsRNA agent which can be processed into asiRNA agent, or a DNA which encodes a dsRNA agent, e.g., a siRNA agent,or precursor thereof). The maintenance dose or doses can be the same orlower than the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 15 mg/kg of body weight per day, e.g., 10,1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. Themaintenance doses are, for example, administered no more than once every2, 5, 10, or 30 days. Further, the treatment regimen may last for aperiod of time which will vary depending upon the nature of theparticular disease, its severity and the overall condition of thepatient. In certain embodiments the dosage may be delivered no more thanonce per day, e.g., no more than once per 24, 36, 48, or more hours,e.g., no more than once for every 5 or 8 days. Following treatment, thepatient can be monitored for changes in his condition and foralleviation of the symptoms of the disease state. The dosage of thecompound may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the composition includes a plurality of dsRNA agentspecies. In another embodiment, the dsRNA agent species has sequencesthat are non-overlapping and non-adjacent to another species withrespect to a naturally occurring target sequence. In another embodiment,the plurality of dsRNA agent species is specific for different naturallyoccurring target genes. In another embodiment, the dsRNA agent is allelespecific.

The dsRNA agents of the invention described herein can be administeredto mammals, particularly large mammals such as nonhuman primates orhumans in a number of ways.

In one embodiment, the administration of the dsRNA agent, e.g., a siRNAagent, composition is parenteral, e.g., intravenous (e.g., as a bolus oras a diffusible infusion), intradermal, intraperitoneal, intramuscular,intrathecal, intraventricular, intracranial, subcutaneous, transmucosal,buccal, sublingual, endoscopic, rectal, oral, vaginal, topical,pulmonary, intranasal, urethral or ocular. Administration can beprovided by the subject or by another person, e.g., a health careprovider. The medication can be provided in measured doses or in adispenser which delivers a metered dose. Selected modes of delivery arediscussed in more detail below.

The invention provides methods, compositions, and kits, for rectaladministration or delivery of dsRNA agents described herein

Methods of Inhibiting Expression of the Target Gene

Embodiments of the invention also relate to methods for inhibiting theexpression of a target gene. The method comprises the step ofadministering the dsRNA agents in any of the preceding embodiments, inan amount sufficient to inhibit expression of the target gene.

Another aspect the invention relates to a method of modulating theexpression of a target gene in a cell, comprising providing to said cella dsRNA agent of this invention. In one embodiment, the target gene isselected from the group consisting of Factor VII, Eg5, PCSK9, TPX2,apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene,GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene,PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepciden,Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene,Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKBgene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene,topoisomerase II alpha gene, mutations in the p73 gene, mutations in thep21(WAF1/CIP1) gene, mutations in the p27(KIP1) gene, mutations in thePPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene,mutations in the MIB I gene, mutations in the MTAI gene, mutations inthe M68 gene, mutations in tumor suppressor genes, and mutations in thep53 tumor suppressor gene.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example 1. In Vitro Screening of siRNA Duplexes

Cell Culture and Transfections:

Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, Va.) were grownto near confluence at 37° C. in an atmosphere of 5% CO₂ in RPMI (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Transfection wascarried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of LipofectamineRNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl ofsiRNA duplexes per well into a 96-well plate and incubated at roomtemperature for 15 minutes. 80 μl of complete growth media withoutantibiotic containing ˜2×10⁴ Hep3B cells were then added to the siRNAmixture. Cells were incubated for either 24 or 120 hours prior to RNApurification. Single dose experiments were performed at 10 nM and 0.1 nMfinal duplex concentration and dose response experiments were done using8, 4 fold serial dilutions with a maximum dose of 10 nM final duplexconcentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl Random primers,0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 1.6 μl of H₂Oper reaction were added into 5 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E (human) Cat #4308313 (rodent)),0.5 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914_ml (human)cat #Rn00562124_ml (rat)) and 5 μl Lightcycler 480 probe master mix(Roche Cat #04887301001) per well in a 384 well plate (Roche cat#04887301001). Real time PCR was done in a Roche LC 480 Real Time PCRmachine (Roche). Each duplex was tested in at least two independenttransfections and each transfection was assayed in duplicate, unlessotherwise noted.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with AD-1955 or naïve cells over the same dose range,or to its own lowest dose. IC₅₀s were calculated for each individualtransfection as well as in combination, where a single IC₅₀ was fit tothe data from both transfections.

The results of gene silencing of the exemplary siRNA duplex with variousmotif modifications of the invention are shown in the table below.

Example 2. RNA Synthesis and Duplex Annealing

1. Oligonucleotide Synthesis:

All oligonucleotides were synthesized on an AKTAoligopilot synthesizeror an ABI 394 synthsizer. Commercially available controlled pore glasssolid support (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramiditeswith standard protecting groups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis unless otherwise specified. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditewere purchased from (Promega). All phosphoramidites were used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichwas used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes was used. The activator was 5-ethyl thiotetrazole(0.75M, American International Chemicals), for the PO-oxidationIodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used.

Ligand conjugated strands were synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcarbohydrate moiety/ligand (for e.g., GalNAc) at the 3′-end of asequence was achieved by starting the synthesis with the correspondingcarbohydrate solid support. Similarly a cholesterol moiety at the 3′-endwas introduced by starting the synthesis on the cholesterol support. Ingeneral, the ligand moiety was tethered to trans-4-hydroxyprolinol via atether of choice as described in the previous examples to obtain ahydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety wasthen coupled to a solid support via a succinate linker or was convertedto phosphoramidite via standard phosphitylation conditions to obtain thedesired carbohydrate conjugate building blocks. Fluorophore labeledsiRNAs were synthesized from the corresponding phosphoramidite or solidsupport, purchased from Biosearch Technologies. The oleyl lithocholic(GalNAc)₃ polymer support made in house at a loading of 38.6 mmol/gram.The Mannose (Man)₃ polymer support was also made in house at a loadingof 42.0 mmol/gram.

Conjugation of the ligand of choice at desired position, for example atthe 5′-end of the sequence, was achieved by coupling of thecorresponding phosphoramidite to the growing chain under standardphosphoramidite coupling conditions unless otherwise specified. Anextended 15 min coupling of 0.1M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid bound oligonucleotide. Oxidation of the internucleotidephosphite to the phosphate was carried out using standard iodine-wateras reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate was introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent The cholesterol phosphoramidite was synthesized in house, andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite was 16 minutes.

2. Deprotection-I (Nucleobase Deprotection)

After completion of synthesis, the support was transferred to a 100 mlglass bottle (VWR). The oligonucleotide was cleaved from the supportwith simultaneous deprotection of base and phosphate groups with 80 mLof a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at55° C. The bottle was cooled briefly on ice and then the ethanolicammonia mixture was filtered into a new 250 ml bottle. The CPG waswashed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume ofthe mixture was then reduced to ˜30 ml by roto-vap. The mixture was thenfrozen on dry ice and dried under vacuum on a speed vac.

3. Deprotection-II (Removal of 2′ TBDMS Group)

The dried residue was resuspended in 26 ml of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionwas then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to6.5, and stored in freezer until purification.

4. Analysis

The oligoncuelotides were analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

5. HPLC Purification

The ligand conjugated oligonucleotides were purified reverse phasepreparative HPLC. The unconjugated oligonucleotides were purified byanion-exchange HPLC on a TSK gel column packed in house. The bufferswere 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mMsodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides were pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotidess werediluted in water to 150 μl and then pipetted in special vials for CGEand LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

6. siRNA Preparation

For the preparation of siRNA, equimolar amounts of sense and antisensestrand were heated in 1×PBS at 95° C. for 5 min and slowly cooled toroom temperature. Integrity of the duplex was confirmed by HPLCanalysis.

TABLE 2 ANGPTL3 modified duplex % of mRNA  Antisense strand (AS)remained conc. Du- Sense strand (S) (SEQ ID NOS 5- (SEQ ID NOS 425-844, of siRNA plex 424, respectively, in order of respectively, in 1 0.10.01 IC50 ID S ID appearance) AS ID order of appearance) nM nM nM (nM)D1000 51000 AfuGfuAfaCfcAfAfGfaGfuAfuUfcCfasu A51000 AfUfgGfaAfuAfcUfcu0.03 0.1 0.47 0.006 uGfgUfuAfcAfusGfsa D1001 51001AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1001 aUfsgGfAfAfuAfcUfc 0.03 0.100.49 0.0065 uuGfgUfuAfcAfusGfsa D1002 51002AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf A51002 aUfgGfAfAfuAfcUfc 0.04 0.100.46 0.0068 uuGfgsUfuAfcAfusGfsa D1003 51003AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf A51003 aUfgGfAfAfuAfcUfcu 0.05 0.120.56 0.0073 uGfgUfsuAfcAfusGfsa D1004 51004 aUGuaACccAGagUAuuCCasuA51004 AUggAAuaCUcuUGguUA 0.07 0.13 0.44 0.008 caUsGsa D1005 51005AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf A51005 aUfgGfAfAfuAfcUfcuu 0.06 0.110.53 0.0093 GfgsUfsuAfcAfusGfsa D1006 51006AfuGfuAfAfccAfAfGfaGfuAfuUfcCfasUf A51006 aUfgGfaAfuAfcUfcuu 0.05 0.160.55 0.0095 GfGfuuAfcAfusGfsa D1007 51007 AfuGfuAfAfCfcAfAfGfaGfuAfuUfcA51007 aUfgGfaAfuAfcUfcuu 0.05 0.14 0.48 0.0098 CfasUf GfguuAfcAfusGfsaD1008 51008 auguaaccaadGadGudAudAcdGasu A51008 aUfgGfaAfuAfcUfca 0.070.11 0.33 0.010 UfuGfgUfuAfcAfusGfs D1009 51009 UfgGfGfAfuUfuCfAfUfgUfaA51009 uCfuugGfuUfaCfaugA 0.03 0.14 0.56 0.0101 AfcCfAfAfgsAffaAfuccCfasUfsc D1010 51010 UfgGfgauUfuCfAfUfgUfaAfcCfaAfgsAf AS1010uCfuUfgGfuUfaCfau 0.03 0.14 0.65 0.0101 gAfaAfUfCfcCfasUfsc D1011 51011aUfGfuAfAfccAfAfGfaGfuAfuUfcCfasUf AS1011 aUfgGfaAfuAfcUfcuuG 0.06 0.100.55 0.011 fGfuuAfcaUfsgsa D1012 51012 UfgGfgAfuUfuCfAfUfgUfaacCfaAfgsAfA51012 uCfuUfgGfUfUfaCfa 0.04 0.13 0.54 0.0114 ugAfaAfuCfcCfasUfsc D101351013 auguaaccaadGadGudAudAcdGasu A51013 aUfgGfaAfuAfcUfcUf 0.11 0.190.49 0.011 ugdGudTadCadTsgsa D1014 51014AfuGfuaaCfcAfAfGfaGfuAfuUfcCfasUf A51014 aUfgGfaAfuAfcUfcuu 0.04 0.160.59 0.013 GfgUfUfAfcAfusGfsa D1015 51015AfuguAfaccAfaGfdAGfdTAdTudCcdAsu A51015 dAUdGgdAadTAfdCUfcU 0.07 0.150.51 0.013 fuGfgUfuAfcAfusGfsa D1016 51016auGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf A51016 aUfgGfaAfuAfcUfcuu 0.05 0.140.64 0.013 GfgUfuAfcAfUfsGfsa D1017 51017 UfGfggAfuUfuCfAfUfgUfAfAA51017 uCfuUfgGfuuaCfaug 0.09 0.41 0.74 0.0133 fcCfaAfgsAfAfaAfuCfCfcasUfsc D1018 51018 AfuguAfaCfcAfAfGfaGfuAfuUfcCfasUf A51018aUfgGfaAfuAfcUfcuu 0.03 0.14 0.61 0.014 GfgUfuAfCfAfusGfsa D1019 51019AfuGfuAfaccAfAfGfaGfuAfuUfcCfasUf A51019 aUfgGfaAfuAfcUfcuuG 0.02 0.20.7 0.014 fGfUfuAfcAfusGfsa D1020 51020AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf A51020 asUfsgGfAfAfuAfcUfc 0.04 0.160.67 0.0156 uuGfgUfuAfcAfusGfsa D1021 51021aUfguAfAfccAfAfgagUfaUfuCfcasUf A51021 aUfGfgAfaUfaCfUfCf 0.11 0.24 0.640.016 uuGfGfuuAfCfaUfsgsa D1022 51022 dTdGggdAdTuudCdAugdTdAacdCdAagsdAAS1022 udCdTugdGdTuadCdAug 0.08 0.27 0.64 0.0161 dAdAaudCdCcasdTsc D102351023 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1023 aUfgsGfAfAfuAfcUfc 0.030.19 0.63 0.0163 uuGfgUfuAfcAfusGfsa D1024 51024UfgGfgAfu UfuCfAfUfguaAfcCfaAfgsAf AS1024 uCfuUfgGfuUfAfCfaug 0.05 0.250.69 0.0164 AfaAfuCfcCfasUfsc D1025 51025 UfgGfgAfuUfuCfAfUfgUfAfAAS1025 uCfuUfgGfuuaCfaug 0.04 0.18 0.75 0.0166 fcCfaAfgsAfAfaAfuCfcCfasUfsc D1026 51026 UfgGfgAfuUfuCfAfUfgUfaAfcCfaAfgsAf AS1026uCfuUfgGfuUfaCfau 0.04 0.19 0.66 0.0178 gAfaAfuCfcCfasUfsc D1027 51027UfgGfgAfuUfuCfAfUfgUfaAfccaAfgsAf AS1027 uCfuUfGfGfuUfaCfaug 0.04 0.190.69 0.018 AfaAfuCfcCfasUfsc D1028 51028dAdTgudAdAccdAdAgadGdTaudTdCcasdT AS1028 adTdGgadAdTacdTdCu 0.15 0.290.72 0.018 udGdGuudAdCausdGsa D1029 51029AdTGdTAdACdCAdAGdAGdTAdTUdCCdAsU AS1029 dAUdGGdAAdTAdCUdCUd 0.1 0.270.61 0.018 TGdGUdTAdCAdTsGsdA D1030 51030UfgGfGfAfuuuCfAfUfgUfaAfcCfaAfgsAf AS1030 uCfuUfgGfuUfaCfaug 0.04 0.210.64 0.0187 AfAfAfuccCfasUfsc D1031 51031AfuGfuAfAfccAfAfGfAfGfuAfuuccAfsu AS1031 AfUfGfGfAfAfuAf 0.06 0.15 0.620.019 CfUfCfUfuGfGf uuAfcAfusGfsa D1032 51032AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1032 asUfgGfAfAfuAfcUfcuu 0.09 0.340.78 0.021 GfgUfsuAfcAfusGfsa D1033 51033UfgGfgAfuUfuCfaUfGfUfaacCfaAfgsAf AS1033 uCfuUfgGfUfUfacaUfg 0.06 0.260.57 0.0212 AfaAfuCfcCfasUfsc D1034 51034AfuGfuAfAfccAfaGfaGfuAfuUfcCfasUf AS1034 aUfgGfaAfuAfcUfcUfu 0.11 0.390.82 0.0216 GfGfuuAfcAfusGfsa D1035 51035UfgGfgAfuuuCfAfUfgUfaAfcCfaAfgsAf AS1035 uCfuUfgGfuUfaCfaug 0.04 0.160.56 0.0222 AfAfAfuCfcCfasUfsc D1036 51036UfgGfGfAfuUfuCfaUfgUfaAfcCfAfAfgsAf AS1036 uCfuugGfuUfaCfaUfgA 0.06 0.310.78 0.0234 faAfuccCfasUfsc D1037 51037UfgGfGfAfuUfuCfAfUfgUfaAfcCfaAfgsAf AS1037 uCfuUfgGfuUfaCfaugA 0.03 0.140.62 0.0235 faAfuccCfasUfsc D1038 51038UfGfggAfUfuuCfAfugUfAfacCfAfagsAf AS1038 uCfUfugGfUfuaCfAfug 0.09 0.390.78 0.0239 AfAfauCfCfcasUfsc D1039 51039AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1039 aUfgGfAfAfuAfcUfcu 0.03 0.140.59 0.025 uGfgUfuAfcAfusGfsa D1040 51040AfuGfuAfaCfcAfAfGfaGfuAfuUfccasUf AS1040 aUfGfGfaAfuAfcUfcu 0.03 0.130.56 0.025 uGfgUfuAfcAfusGfsa D1041 51041AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1041 asUfgGfAfAfuAfcUfc 0.06 0.270.79 0.0252 uuGfgUfuAfcAfusGfsa D1042 51042UfgGfgAfuuuCfAfUfgUfAfAfcCfaAfgsAf AS1042 uCfuUfgGfuuaCfaugAf 0.05 0.270.67 0.0259 AfAfuCfcCfasUfsc D1043 51043AfuGfuAfaCfcAfAfGfaGfuauUfcCfasUf AS1043 aUfgGfaAfUfAfcUfcuu 0.02 0.160.63 0.027 GfgUfuAfcAfusGfsa D1044 51044AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1044 asUfgGfAfAfuAfcUfcu 0.06 0.300.81 0.0271 uGfgsUfsuAfcAfusGfsa D1045 51045aUfguAfAfccAfAfgaGfGfauUfCfcasUf AS1045 aUfGfgaAfUfacUfCfuu 0.12 0.290.8 0.028 GfGfuuAfCfaUfsgsa D1046 51046AfuGfuAfaCfcAfAfGfaguAfuUfcCfasUf AS1046 aUfgGfaAfuAfCfUfcuu 0.03 0.150.59 0.030 GfgUfuAfcAfusGfsa D1047 51047UfgGfGfAfuUfuCfaUfgUfAfAfcCfaAfgsAf AS1047 uCfuUfgGfuuaCfaUfg 0.08 0.440.83 0.0324 AfaAfuccCfasUfsc D1048 51048AfuGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf AS1048 aUfgGfaAfuAfcUfcu 0.07 0.230.67 0.036 uGfgUfuAfcAfusGfsa D1049 51049AfuGfuAfAfccAfAfGfAfGfuAfuuccAfsu AS1049 AfUfGfGfAfAfuAf 0.08 0.23 0.730.037 CfUfCfUfUfGfGfU fuAfCfAfusGfsa D1050 51050UfgGfgAfuuuCfaUfgUfaAfcCfAfAfgsAf AS1050 uCfuugGfuUfaCfaUfg 0.06 0.290.78 0.0372 AfAfAfuCfcCfasUfsc D1051 51051 AfuGfuAfaccaagaguAfuUfcCfasUfAS1051 aUfgGfaAfudAcdTcdT 0.12 0.41 0.86 0.040 udGgdTuAfcAfusgsa D105251052 AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu AS1052 aUfgGfaAfuAfcUfcUf 0.10.22 0.72 0.042 uGfgUfuAfcAfusGfsa D1053 51053AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu AS1053 dAUdGGdAAfuAfcUfcUfu 0.09 0.310.69 0.044 GfGfUfuAfCfAfusGfsa D1054 51054AfuGfuAfaCfcAfaGfadGdTAfuUfcdCdAsUf AS1054 adTdGGfaAfudAdCUfcU 0.1 0.450.75 0.047 fuGfgUfuAfcAfusGfsa D1055 51055AfuguAfaccAfaGfaGfdTAdTUdCcdAsu AS1055 dAUdGGdAadTAfcUfcUf 0.12 0.26 0.70.049 uGfgUfuAfcAfusGfsa D1056 51056 AuGuAaCcAaGaGuAuUcCasU AS1056aUgGaAuAcUcUuGgU 0.08 0.24 0.65 0.050 uAcAusGsa D1057 51057AfuguAfaccAfagaGfuauUfccasUf AS1057 aUfGfGfaAfUfAfcUfCfUf 0.14 0.42 0.620.051 uGfGfUfuAfCfAfusGfsa D1058 51058 AfuGfuAfaccaagaguAfuUfcCfasUfAS1058 aUfgGfaAfudAcdTcdTu 0.12 0.36 0.86 0.053 dGgdTuAfcAfusGfsa D105951059 AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu AS1059 dAUdGGdAadTAfdCUfcUfu 0.090.27 0.7 0.054 GfgUfuAfcAfusGfsa D1060 51060adTgudAdAccdAdAgagdTadTudCcasdT AS1060 adTdGgdAadTadCdTdC 0.11 0.37 0.660.056 uudGdGuudAdCadTsgsa D1061 51061 AfuGfuAfaCfcAfaGfdAdG AS1061adTdGGfaAfuAfdCdTcU 0.1 0.31 0.77 0.059 uAfuUfcdCdAsUffuGfgUfuAfcAfusGfsa D1062 51062 AfuguAfaccAfaGfdAGfdTAdTudCcdAsu AS1062aUfgGfaAfuAfcUfcUf 0.1 0.27 0.65 0.059 uGfgUfuAfcAfusGfsa D1063 51063adTdGuadAdCccdAdGagdTdAuudCdCasu AS1063 dAdTggdAdAuadCdTcu 0.12 0.440.82 0.064 dTdGgudTdAcadTsdGsa D1064 51064AfuGfuAfaCfcAfaGfaGfdTdAuUfcdC AS1064 adTdGGfaAfdTdAcUfcU 0.12 0.32 0.830.064 dAsUf fuGfgUfuAfcAfusGfsa D1065 51065AfuguAfaccAfaGfaGfdTAdTudCcdAsu AS1065 dAUdGgdAadTAfcUfcU 0.13 0.34 0.720.066 fuGfgUfuAfcAfusGfsa D1066 51066 AfuGfuAfaCfcAfaGfaGfudAdTUf AS1066adTdGGfadAdTAfcUfcU 0.11 0.33 0.72 0.067 cdCdAsUf fuGfgUfuAfcAfusGfsaD1067 51067 AfuguAfaccAfaGfaGfdTAdTUdCcdAsu AS1067 aUfgGfaAfuAfcUfcUf0.11 0.37 0.62 0.070 uGfgUfuAfcAfusGfsa D1068 51068AfuguAfaccAfaGfaGfdTAdTUdCcdAsu AS1068 dAUdGGdAAuAfcUfcUfu 0.16 0.330.64 0.072 GfGfUfuAfCfAfusGfsa D1069 51069aUfGfuaAfCfccAfGfagUfAfuuCfCfasu AS1069 AfUfggAfAfuaCfUfcuU 0.14 0.430.73 0.074 fGfguUfAfcaUfsGfsa D1070 51070AfuGfuAfaCfCfAfaGfaguAfuUfcCfasUf AS1070 aUfgGfaAfuAfCfUfcU 0.08 0.420.94 0.075 fuggUfuAfcAfusGfsa D1071 51071UfgGfgAfuuuCfaUfgUfaAfcCfaAfgsAf AS1071 uCfuUfgGfuUfaCfaUf 0.14 0.280.83 0.0797 gAfAfAfuCfcCfasUfsc D1072 51072AfuGfuAfaCfcAfaGfAfGfuauUfcCfasUf AS1072 aUfgGfaAfUfAfcucUf 0.05 0.260.8 0.082 uGfgUfuAfcAfusGfsa D1073 51073 AfuGfuAfaCfcAfaGfadGdT AS1073aUfgGfadAdTdAdCUfc 0.12 0.41 0.73 0.083 dAdTUfcCfasUfUfuGfgUfuAfcAfusGfsa D1074 51074 AfUfguAfAfccAfAfgaGfUfauUfCfcasUfAS1074 aUfGfgaAfUfacUfCf 0.14 0.44 0.75 0.086 uuGfGfuuAfCfausGfsa D107551075 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1075 aUfgGfdAdAdTdAcUfcU 0.10.41 0.72 0.088 fuGfgUfuAfcAfusGfsa D1076 51076AfuGfuAfaCfcAfaGfaGfudAdT AS1076 aUfgdGdAdAdTAfcUfcU 0.15 0.45 0.860.088 dTdCCfasUf fuGfgUfuAfcAfusGfsa D1077 51077AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasu AS1077 AfUfgGfaAfuAfcUfcU 0.08 0.460.95 0.092 fuGfgUfuAfcAfusGfsa D1078 51078AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1078 dAUdGGdAadTAfcUfcU 0.09 0.320.76 0.093 fuGfgUfuAfcAfusGfsa D1079 51079AfuguAfaccAfaGfaGfdTadTudCcdAsu AS1079 dAudGgdAadTAfcUfcUf 0.14 0.380.76 0.095 uGfgUfuAfcAfusGfsa D1080 51080AfuGfuAfaCfcAfaGfAfGfuAfuucCfasUf AS1080 aUfgGfAfAfuAfcucU 0.05 0.420.86 0.099 fuGfgUfuAfcAfusGfsa D1081 51081 AfuGfuAfaCfcAfaGfaGfu AS1081dAdTdGdGaAfuAfcUfc 0.17 0.47 0.9 0.105 AfuUfdCdCdAsdTUfuGfgUfuAfcAfusGfsa D1082 51082 AfuGfuAfaccaagaguAfuUfcCfasUf AS1082aUfgGfaAfudACfudCU 0.12 0.44 0.83 0.106 fudGGfudTAfcAfusgsa D1083 51083AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1083 adTdGGfaAfdTdAcUfcU 0.11 0.340.74 0.109 fuGfgUfuAfcAfusGfsa D1084 51084AfuGfuAfAfCfcAfaGfaGfuauUfcCfasUf AS1084 aUfgGfaAfUfAfcUfcUf 0.1 0.450.93 0.117 uGfguuAfcAfusGfsa D1085 51085AfuGfUfAfaCfcAfaGfaGfuauUfcCfasUf AS1085 aUfgGfaAfUfAfcUfcUf 0.07 0.420.78 0.120 uGfgUfuacAfusGfsa D1086 51086aUfguAfAfccAfAfgaGfuAfuUfcCfasUf AS1086 aUfgGfaAfuAfcUfCfuu 0.17 0.450.83 0.1197 GfGfuuAfCfaUfsgsa D1087 51087AfuGfuAfaCfcAfaGfaGfUfAfuUfcCfasu AS1087 AfUfgGfaAfuacUfcUfu 0.05 0.30.7 0.120 GfgUfuAfcAfusGfsa D1088 51088AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1088 aUfgGfaAfuAfcUfcUf 0.11 0.460.8 0.120 uGfgUfuAfcAfusgsa D1089 51089AfuGfuAfaCfcAfaGfaGfUfAfuUfcCfasUf AS1089 aUfgGfaAfuacUfcUfu 0.14 0.490.85 0.122 GfgUfuAfcAfusGfsa D1090 51090AfuGfuAfaCfcAfaGfaGfuauUfcCfasUf AS1090 aUfgGfaAfUfAfcUfcU 0.1 0.41 0.850.125 fuGfgUfuAfcAfusGfsa D1091 51091 AfuguAfaccAfaGfaGfdTAdTudCcdAsuAS1091 aUfgGfaAfuAfcUfcUf 0.16 0.38 0.77 0.125 uGfgUfuAfcAfusGfsa D109251092 AfuGfuAfaCfcAfaGfAfGfuAfuUfcCfasu AS1092 AfUfgGfaAfuAfcucUf 0.050.31 0.93 0.126 uGfgUfuAfcAfusGfsa D1093 51093auGfuAfaCfcAfaGfAfGfuAfuUfcCfasUf AS1093 aUfgGfaAfuAfcucUfu 0.06 0.330.9 0.135 GfgUfuAfcAfUfsGfsa D1094 51094AfuGfuAfaCfcAfaGfaGfUfAfuUfccasUf AS1094 aUfGfGfaAfuacUfcUf 0.07 0.390.85 0.142 uGfgUfuAfcAfusGfsa D1095 51095AfuGfuAfaCfcAfaGfAfGfuAfuUfcCfasUf  AS1095 aUfgGfaAfuAfcucUfu 0.09 0.390.76 0.146 GfgUfuAfcAfusGfsa D1096 51096AfuGfuAfaCfcAfaGfaGfUfAfuucCfasUf AS1096 aUfgGfAfAfuacUfcUf 0.06 0.380.85 0.147 uGfgUfuAfcAfusGfsa D1097 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uUaUAgaGCaaGAacACu 0.13 0.31 0.76 gUUsusuD1410 51410 AfaCfaAfuGfuUfcUfuGfcAfdCdTdA AS1410 udTdAdTdAdGaGfcAfaGfa0.77 0.94 0.93 dTdAsAf GfcAfcAfgUfusUfsu D1411 51411aacAfgugUfucuUfgcuCfuauAfsa AS1411 uUfaUfAfgAfGfCfaAfGf 0.23 0.53 1.04AfaCfAfCfuGfUfUfsusu D1412 51412 aacdAgugdTucudTgcudCuaudAsa AS1412udTadTdAgdAdGdCadAdGd 0.30 0.64 0.90 AadCdAdCudGdTdTsusu D1413 51413AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasa AS1413 UfUfaUfaGfaGfcAfaGfaA 0.09 0.190.63 fcAfcUfgUfusUfsu D1414 51414 AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasaAS1414 UfUfaUfaGfaGfcAfaGfaa 0.11 0.28 0.66 cAfcUfgUfusUfsu D1415 51415AfaCfaGfuGfuUfcUfuGfCfUfcUfaUfasa AS1415 UfUfaUfaGfagcAfaGfaA 0.06 0.130.53 fcAfcUfgUfusUfsu D1416 51416 aacaguguucuugcucuauasa AS1416UfUfAfUfAfGfAfG 0.20 0.53 0.99 fCfAfAfGfAfAfCfAfC fUfGfUfUfsusu D141751417 AfaCfaGfuGfuUfcUfuGfcUfcUfAfUfasa AS1417 UfUfauaGfaGfcAfaGfaAf0.07 0.17 0.53 cAfcUfgUfusUfsu D1418 51418aAfCfagUfGfuuCfUfugCfUfcuAfUfasa AS1418 UfUfauAfGfagCfAfagAf 0.08 0.200.70 AfcaCfUfguUfsUfsu D1419 51419 AfaCfAfGfuGfuUfcUfuGfcUfcU AS1419uUfaUfaGfaGfcAfaGfa 0.08 0.20 0.70 faUfasAf AfcAfcugUfusUfsUf

Example 3. In Vitro Silencing Activity with Various ChemicalModifications on TTR siRNA

The IC₅₀ for each modified siRNA is determined in Hep3B cells bystandard reverse transfection using Lipofectamine RNAiMAX. In brief,reverse transfection is carried out by adding 5 μl of Opti-MEM to 5 μlof siRNA duplex per well into a 96-well plate along with 10 μl ofOpti-MEM plus 0.5 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) and incubating at room temperature for15-20 minutes. Following incubation, 100 μl of complete growth mediawithout antibiotic containing 12,000-15,000 Hep3B cells is then added toeach well. Cells are incubated for 24 hours at 37° C. in an atmosphereof 5% CO2 prior to lysis and analysis of ApoB and GAPDH mRNA by bDNA(Quantigene). Seven different siRNA concentrations ranging from 10 nM to0.6 μM are assessed for IC₅₀ determination and ApoB/GAPDH for ApoBtransfected cells is normalized to cells transfected with 10 nM LucsiRNA.

Abbreviation Nucleotide(s) Af 2′-F-adenosine Cf 2′-F-cytidine Gf2′-F-guanosine Uf 2′-F-uridine A adenosine C cytidine G guanosine Uuridine a 2′-O-methyladenosine c 2′-O-methylcytidine g2′-O-methylguanosine u 2′-O-methyluridine dT 2′-deoxythymidine sphosphorothioate linkage

TABLE 3 ANGPTL3 modified duplex SS seq (SEQ ID NOS 845- RNAimax, Hep3bDuplex Sense 1025, respectively, in order AS seq (SEQ ID NOS 1026-1206,  10 0.1 0.025 ID ID of appearance) AS IDrespectively, in order of appearance) nM nM nM D2000 S2000UfcAfcAfaUfuAfAfGfcUfcCfuUfcUfuUf A2000aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc 0.036 0.274 0.233 D2001 S2001UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUf A2001aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa 0.044 0.278 0.247 D2002 S2002GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAf A2002uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa 0.062 0.474 0.449 D2003 S2003GfgAfcAfuGfgUfCfUfuAfaAfgAfcUfuUf A2003aAfaGfuCfuUfuAfagaCfcAfuGfuCfcsCfsa 0.303 1.042 0.912 D2004 S2004CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUf A2004aUfcAfaAfuAfuGfuugAfgUfuUfuUfgsAfsa 0.102 0.623 0.499 D2005 S2005AfcCfaGfuGfaAfAfUfcAfaAfgAfaGfaAf A2005uUfcUfuCfuUfuGfauuUfcAfcUfgGfusUfsu 0.124 0.901 0.756 D2006 S2006CfaCfaAfuUfaAfGfCfuCfcUfuCfuUfuUf A2006aAfaAfgAfaGfgAfgcuUfaAfuUfgUfgsAfsa 0.069 0.269 0.244 D2007 S2007CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAf A2007uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa 0.052 0.622 0.589 D2008 S2008UfcAfaCfaUfaUfUfUfgAfuCfaGfuCfuUf A2008aAfgAfcUfgAfuCfaaaUfaUfgUfuGfasGfsu 0.133 0.798 0.785 D2009 S2009AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAf A2009uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc 0.097 0.671 0.528 D2010 S2010AfcAfaUfuAfaGfCfUfcCfuUfcUfuUfuUf A2010aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa 0.145 0.308 0.293 D2011 S2011CfuCfcAfgAfgCfCfAfaAfaUfcAfaGfaUf A2011aUfcUfuGfaUfuUfuggCfuCfuGfgAfgsAfsu 0.122 0.882 0.938 D2012 S2012CfgAfuGfuAfaAfAfAfuUfuUfaGfcCfaAf A2012uUfgGfcUfaAfaAfuuuUfuAfcAfuCfgsUfsc 0.102 0.843 0.733 D2013 S2013GfuCfuUfaAfaGfAfCfuUfuGfuCfcAfuAf A2013uAfuGfgAfcAfaAfgucUfuUfaAfgAfcsCfsa 1.133 1.105 1.022 D2014 S2014CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUf A2014aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg 0.077 0.413 0.450 D2015 S2015AfcUfgAfgAfaGfAfAfcUfaCfaUfaUfaAf A2015uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc 0.055 0.293 0.364 D2016 S2016CfcAfgAfgCfcAfAfAfaUfcAfaGfaUfuUf A2016aAfaUfcUfuGfaUfuuuGfgCfuCfuGfgsAfsg 0.080 0.650 0.499 D2017 S2017GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUf A2017aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu 0.076 0.605 0.579 D2018 S2018UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAf A2018uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc 1.326 1.098 0.927 D2019 S2019AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUf A2019aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa 0.047 0.560 0.477 D2020 S2020CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAf A2020uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu 0.066 0.690 0.681 D2021 S2021AfaUfuAfaGfcUfCfCfuUfcUfuUfuUfaUf A2021aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu 0.041 0.611 0.251 D2022 S2022AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAf A2022uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg 0.053 0.555 0.516 D2023 S2023UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAf A2023uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg 0.779 1.045 0.963 D2024 S2024GfgGfcCfaAfaUfUfAfaUfgAfcAfuAfuUf A2024aAfuAfuGfuCfaUfuaaUfuUfgGfcCfcsUfsu 1.487 0.949 0.883 D2025 S2025AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf A2025aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg 0.043 0.432 0.477 D2026 S2026AfgAfaCfuAfcAfUfAfuAfaAfcUfaCfaAf A2026uUfgUfaGfuUfuAfuauGfuAfgUfuCfusUfsc 0.324 1.042 0.905 D2027 S2027AfuUfaAfgCfuCfCfUfuCfuUfuUfuAfuUf A2027aAfuAfaAfaAfgAfaggAfgCfuUfaAfusUfsg 0.042 0.283 0.224 D2028 S2028AfgAfuUfuGfcUfAfUfgUfuAfgAfcGfaUf A2028aUfcGfuCfuAfaCfauaGfcAfaAfuCfusUfsg 0.349 0.936 0.896 D2029 S2029UfcAfgUfuGfgGfAfCfaUfgGfuCfuUfaAf A2029uUfaAfgAfcCfaUfgucCfcAfaCfuGfasAfsg 0.914 0.907 0.944 D2030 S2030GfgCfcAfaAfuUfAfAfuGfaCfaUfaUfuUf A2030aAfaUfaUfgUfcAfuuaAfuUfuGfgCfcsCfsu 0.047 0.353 0.326 D2031 S2031CfaUfaUfuUfgAfUfCfaGfuCfuUfuUfuAf A2031uAfaAfaAfgAfcUfgauCfaAfaUfaUfgsUfsu 0.110 0.867 0.842 D2032 S2032UfaCfaUfaUfaAfAfCfuAfcAfaGfuCfaAf A2032uUfgAfcUfuGfuAfguuUfaUfaUfgUfasGfsu 0.200 0.699 0.656 D2033 S2033UfuUfuAfuUfgUfUfCfcUfcUfaGfuUfaUf A2033aUfaAfcUfaGfaGfgaaCfaAfuAfaAfasAfsg 0.050 0.218 0.192 D2034 S2034UfuGfcUfaUfgUfUfAfgAfcGfaUfgUfaAf A2034uUfaCfaUfcGfuCfuaaCfaUfaGfcAfasAfsu 0.096 0.792 0.640 D2035 S2035CfaGfuUfgGfgAfCfAfuGfgUfcUfuAfaAf A2035uUfuAfaGfaCfcAfuguCfcCfaAfcUfgsAfsa 0.127 0.936 0.890 D2036 S2036AfaAfuUfaAfuGfAfCfaUfaUfuUfcAfaAf A2036uUfuGfaAfaUfaUfgucAfuUfaAfuUfusGfsg 0.061 0.683 0.668 D2037 S2037GfaUfcAfgUfcUfUfUfuUfaUfgAfuCfuAf A2037uAfgAfuCfaUfaAfaaaGfaCfuGfaUfcsAfsa 0.157 1.010 0.723 D2038 S2038AfcAfuAfuAfaAfCfUfaCfaAfgUfcAfaAf A2038uUfuGfaCfuUfgUfaguUfuAfuAfuGfusAfsg 0.047 0.532 0.525 D2039 S2039UfuUfaUfuGfuUfCfCfuCfuAfgUfuAfuUf A2039aAfuAfaCfuAfgAfggaAfcAfaUfaAfasAfsa 0.031 0.505 0.238 D2040 S2040UfgCfuAfuGfuUfAfGfaCfgAfuGfuAfaAf A2040uUfuAfcAfuCfgUfcuaAfcAfuAfgCfasAfsa 0.056 0.484 0.408 D2041 S2041GfgGfaCfaUfgGfUfCfuUfaAfaGfaCfuUf A2041aAfgUfcUfuUfaAfgacCfaUfgUfcCfcsAfsa 0.570 0.999 0.994 D2042 S2042UfgAfcAfuAfuUfUfCfaAfaAfaCfuCfaAf A2042uUfgAfgUfuUfuUfgaaAfuAfuGfuCfasUfsu 0.065 0.870 0.728 D2043 S2043AfuCfaGfuCfuUfUfUfuAfuGfaUfcUfaUf A2043aUfaGfaUfcAfuAfaaaAfgAfcUfgAfusCfsa 0.048 0.362 0.282 D2044 S2044CfaUfaUfaAfaCfUfAfcAfaGfuCfaAfaAf A2044uUfuUfgAfcUfuGfuagUfuUfaUfaUfgsUfsa 0.314 0.904 0.937 D2045 S2045CfuUfgAfaCfuCfAfAfcUfcAfaAfaCfuUf A2045aAfgUfuUfuGfaGfuugAfgUfuCfaAfgsUfsg 0.060 0.295 0.251 D2046 S2046CfuAfcUfuCfaAfCfAfaAfaAfgUfgAfaAf A2046uUfuCfaCfuUfuUfuguUfgAfaGfuAfgsAfsa 0.052 0.570 0.599 D2047 S2047AfaGfaGfcAfaCfUfAfaCfuAfaCfuUfaAf A2047uUfaAfgUfuAfgUfuagUfuGfcUfcUfusCfsu 0.028 0.369 0.381 D2048 S2048AfaAfcAfaGfaUfAfAfuAfgCfaUfcAfaAf A2048uUfuGfaUfgCfuAfuuaUfcUfuGfuUfusUfsu 0.039 0.227 0.204 D2049 S2049GfcAfuAfgUfcAfAfAfuAfaAfaGfaAfaUf A2049aUfuUfcUfuUfuAfuuuGfaCfuAfuGfcsUfsg 0.032 0.437 0.422 D2050 S2050AfuAfuAfaAfcUfAfCfaAfgUfcAfaAfaAf A2050uUfuUfuGfaCfuUfguaGfuUfuAfuAfusGfsu 0.297 0.946 0.850 D2051 S2051GfaAfcUfcAfaCfUfCfaAfaAfcUfuGfaAf A2051uUfcAfaGfuUfuUfgagUfuGfaGfuUfcsAfsa 0.179 0.929 0.884 D2052 S2052UfaCfuUfcAfaCfAfAfaAfaGfuGfaAfaUf A2052aUfuUfcAfcUfuUfuugUfuGfaAfgUfasGfsa 0.091 0.536 0.524 D2053 S2053AfgAfgCfaAfcUfAfAfcUfaAfcUfuAfaUf A2053aUfuAfaGfuUfaGfuuaGfuUfgCfuCfusUfsc 0.086 0.611 0.621 D2054 S2054GfaUfaAfuAfgCfAfUfcAfaAfgAfcCfuUf A2054aAfgGfuCfuUfuGfaugCfuAfuUfaUfcsUfsu 0.058 0.676 0.591 D2055 S2055CfaUfaGfuCfaAfAfUfaAfaAfgAfaAfuAf A2055uAfuUfuCfuUfuUfauuUfgAfcUfaUfgsCfsu 0.048 0.630 0.674 D2056 S2056UfaUfaAfaCfuAfCfAfaGfuCfaAfaAfaUf A2056aUfuUfuUfgAfcUfuguAfgUfuUfaUfasUfsg 0.072 0.534 0.459 D2057 S2057AfaCfuCfaAfcUfCfAfaAfaCfuUfgAfaAf A2057uUfuCfaAfgUfuUfugaGfuUfgAfgUfusCfsa 0.161 0.864 0.775 D2058 S2058AfcUfuCfaAfcAfAfAfaAfgUfgAfaAfuAf A2058uAfuUfuCfaCfuUfuuuGfuUfgAfaGfusAfsg 0.198 0.969 0.865 D2059 S2059GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUf A2059aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu 0.031 0.253 0.210 D2060 S2060AfaCfcAfaCfaGfCfAfuAfgUfcAfaAfuAf A2060uAfuUfuGfaCfuAfugcUfgUfuGfgUfusUfsa 0.035 0.561 0.569 D2061 S2061AfgUfcAfaAfuAfAfAfaGfaAfaUfaGfaAf A2061uUfcUfaUfuUfcUfuuuAfuUfuGfaCfusAfsu 0.057 0.668 0.386 D2062 S2062AfgUfcAfaAfaAfUfGfaAfgAfgGfuAfaAf A2062uUfuAfcCfuCfuUfcauUfuUfuGfaCfusUfsg 0.720 1.017 0.924 D2063 S2063CfuUfgAfaAfgCfCfUfcCfuAfgAfaGfaAf A2063uUfcUfuCfuAfgGfaggCfuUfuCfaAfgsUfsu 0.324 1.020 0.963 D2064 S2064CfuUfcAfaCfaAfAfAfaGfuGfaAfaUfaUf A2064aUfaUfuUfcAfcUfuuuUfgUfuGfaAfgsUfsa 0.048 0.549 0.531 D2065 S2065CfaAfcUfaAfcUfAfAfcUfuAfaUfuCfaAf A2065uUfgAfaUfuAfaGfuuaGfuUfaGfuUfgsCfsu 0.046 0.739 0.649 D2066 S2066AfcCfaAfcAfgCfAfUfaGfuCfaAfaUfaAf A2066uUfaUfuUfgAfcUfaugCfuGfuUfgGfusUfsu 0.076 0.840 0.777 D2067 S2067GfaAfcCfcAfcAfGfAfaAfuUfuCfuCfuAf A2067uAfgAfgAfaAfuUfucuGfuGfgGfuUfcsUfsu 0.103 0.916 0.808 D2068 S2068GfaAfuAfuGfuCfAfCfuUfgAfaCfuCfaAf A2068uUfgAfgUfuCfaAfgugAfcAfuAfuUfcsUfsu 0.046 0.532 0.S20 D2069 S2069UfgAfaAfgCfcUfCfCfuAfgAfaGfaAfaAf A2069uUfuUfcUfuCfuAfggaGfgCfuUfuCfasAfsg 0.067 0.894 0.822 D2070 S2070UfuCfaAfcAfaAfAfAfgUfgAfaAfuAfuUf A2070aAfuAfuUfuCfaCfuuuUfuGfuUfgAfasGfsu 0.052 0.557 0.395 D2071 S2071AfaCfuAfaCfuAfAfCfuUfaAfuUfcAfaAf A2071uUfuGfaAfuUfaAfguuAfgUfuAfgUfusGfsc 0.025 0.220 0.232 D2072 S2072CfcAfaCfaGfcAfUfAfgUfcAfaAfuAfaAf A2072uUfuAfuUfuGfaCfuauGfcUfgUfuGfgsUfsu 0.293 0.923 0.899 D2073 S2073AfaCfcCfaCfaGfAfAfaUfuUfcUfcUfaUf A2073aUfaGfaGfaAfaUfuucUfgUfgGfgUfusCfsu 0.021 0.375 0.356 D2074 S2074UfgUfcAfcUfuGfAfAfcUfcAfaCfuCfaAf A2074uUfgAfgUfuGfaGfuucAfaGfuGfaCfasUfsa 0.052 0.402 0.513 D2075 S2075GfaAfaGfcCfuCfCfUfaGfaAfgAfaAfaAf A2075uUfuUfuCfuUfcUfaggAfgGfcUfuUfcsAfsa 0.171 0.904 0.893 D2076 S2076AfaUfaUfuUfaGfAfAfgAfgCfaAfcUfaAf A2076uUfaGfuUfgCfuCfuucUfaAfaUfaUfusUfsc 0.142 0.614 0.688 D2077 S2077AfcUfaAfcUfaAfCfUfuAfaUfuCfaAfaAf A2077uUfuUfgAfaUfuAfaguUfaGfuUfaGfusUfsg 0.020 0.312 0.316 D2078 S2078CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAf A2078uUfuUfaUfuUfgAfcuaUfgCfuGfuUfgsGfsu 0.026 0.313 0.393 D2079 S2079CfcAfcAfgAfaAfUfUfuCfuCfuAfuCfuUf A2079aAfgAfuAfgAfgAfaauUfuCfuGfuGfgsGfsu 0.012 0.596 0.345 D2080 S2080GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAf A2080uUfuGfaGfuUfgAfguuCfaAfgUfgAfcsAfsu 0.054 0.503 0.456 D2081 S2081CfuCfcUfaGfaAfGfAfaAfaAfaUfuCfuAf A2081uAfgAfaUfuUfuUfucuUfcUfaGfgAfgsGfsc 0.050 0.596 0.531 D2082 S2082AfuUfuAfgAfaGfAfGfcAfaCfuAfaCfuAf A2082uAfgUfuAfgUfuGfcucUfuCfuAfaAfusAfsu 0.064 0.806 0.928 D2083 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S2178UfaCfuCfuAfuAfAfAfaUfcAfaCfcAfaAf A2178uUfuGfgUfuGfaUfuuuAfuAfgAfgUfasUfsa 0.063 0.897 0.869 D2179 S2179GfaAfcUfgAfgGfCfAfaAfuUfuAfaAfaAf A2179uUfuUfuAfaAfuUfugcCfuCfaGfuUfcsAfsu 0.178 0.858 0.869 D2180 S2180CfaGfaGfuAfuGfUfGfuAfaAfaAfuCfuUf A2180aAfgAfuUfuUfuAfcacAfuAfcUfcUfgsUfsg 0.436 0.677 0.813

Example 4. In Vitro Silencing Activity with Various ChemicalModifications on ANGPTL3 siRNA

Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in RPMI (ATCC) supplemented with 10% FBS,streptomycin, and glutamine (ATCC) before being released from the plateby trypsinization. Transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2×10⁴ Hep3Bcells were then added to the siRNA mixture. Cells were incubated foreither 24 or 120 hours prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM final duplexconcentration and dose response experiments were done at 10, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nMfinal duplex concentration unless otherwise stated.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR

2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E), 0.5 μl ANGPTL TaqMan probe(Applied Biosystems cat #Hs00205581_m1) and 5 μl Lightcycler 480 probemaster mix (Roche Cat #04887301001) per well in a 384 well 50 plates(Roche cat #04887301001). Real time PCR was done in an ABI 7900HT RealTime PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Eachduplex was tested in two independent transfections, and eachtransfection was assayed in duplicate, unless otherwise noted in thesummary tables.

To calculate relative fold change, real time data was analyzed using theΔΔCt method and normalized to assays performed with cells transfectedwith 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculatedusing a 4 parameter fit model using XLFit and normalized to cellstransfected with AD-1955 or naïve cells over the same dose range, or toits own lowest dose. AD-1955 sequence, used as a negative control,targets luciferase and has the following sequence:

(SEQ ID NO: 1207) sense: cuuAcGcuGAGuAcuucGAdTsdT; (SEQ ID NO: 1208)antisense: UCGAAGuACUcAGCGuAAGdTsdT.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

We claim:
 1. A double-stranded RNAi agent capable of inhibiting theexpression of a target gene, comprising a sense strand and an antisensestrand, each strand having 14 to 30 nucleotides, wherein the duplex isrepresented by formula (III): sense: 5′n_(p) -N_(a) -(X X X) _(i)-N_(b) -Y Y Y -N_(b) -(Z Z Z)_(j) -N_(a) -n_(q) 3′ antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein: i, j, k, and l are each independently 0 or 1, provided that atleast one of i, j, k, and l is 1; p and q are each independently 0-6;each Na and Na′ independently represents an oligonucleotide sequencecomprising 2-20 nucleotides which are either modified or unmodified orcombinations thereof, each sequence comprising at least two differentlymodified nucleotides, each Nb and Nb′ independently represents anoligonucleotide sequence comprising 1-10 modified nucleotides; eachn_(p), n_(p)′, n_(q) and n_(q)′ independently represents an overhangnucleotide sequence comprising 0-6 nucleotides; and XXX, YYY, ZZZ,X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides; andwherein the modification on Nb is different than the modification on Yand the modification on Nb′ is different than the modification on Y′. 2.The double-stranded RNAi agent of claim 1, wherein i is 1; j is 1; orboth i and j are
 1. 3. The double-stranded RNAi agent of claim 1,wherein k is 1; l is 1; or both k and l are
 1. 4. The double-strandedRNAi agent of claim 1, wherein the YYY motif occurs at or near thecleavage site of the sense strand.
 5. The double-stranded RNAi agent ofclaim 1, wherein the Y′Y′Y′ motif occurs at the 11, 12 and 13 positionsof the antisense strand from the 5′-end.
 6. The double-stranded RNAiagent of claim 5, wherein the Y′ is 2′-OMe.
 7. The double-stranded RNAiagent of claim 1, wherein formula (III) is represented as formula(IIIa): 5′ n_(p) -N_(a) -Y Y Y -N_(b) -Z Z Z -N_(a) -n_(q) 3′ 3′n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′-n_(q)′ 5′ (IIIa)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.
 8. Thedouble-stranded RNAi agent of claim 1, wherein formula (III) isrepresented as formula (IIIb): 5′n_(p)-N_(a)-X X X -N_(b)-Y Y Y -N_(a)-n_(q) 3′ 3′n_(p)-N_(a)-X′X′X′-N_(b)-Y′Y′Y′-N_(a)-n_(q) 5′ (IIIb)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.
 9. Thedouble-stranded RNAi agent of claim 1, wherein formula (III) isrepresented as formula (IIIc): 5′n_(p)-N_(a)-X X X -N_(b)-Y Y Y -N_(b)-Z Z Z -N_(a)-n_(q) 3′ 3′n_(p)-N_(a)-X′X′X′-N_(b)-Y′Y′Y′-N_(b)-Z′Z′Z′-N_(a)-n_(q) 5′ (IIIc)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-10 modified nucleotides.
 10. The double-stranded RNAi agentof claim 1, wherein the duplex region is 17-30 nucleotide pairs inlength.
 11. The double-stranded RNAi agent of claim 10, wherein theduplex region is 17-19 nucleotide pairs in length.
 12. Thedouble-stranded RNAi agent of claim 10, wherein the duplex region is27-30 nucleotide pairs in length.
 13. The double-stranded RNAi agent ofclaim 1, wherein each strand has 17-30 nucleotides.
 14. Thedouble-stranded RNAi agent of claim 1, wherein the modifications on thenucleotides are selected from the group consisting of LNA, HNA, CeNA,2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro,2′-deoxy, and combinations thereof.
 15. The double-stranded RNAi agentof claim 14, wherein the nucleotides are modified with either 2′-OCH₃ or2′-F.
 16. The double-stranded RNAi agent of claim 1, further comprisingat least one ligand.
 17. The double-stranded RNAi agent of claim 16,wherein the ligand is a one or more GalNAc derivatives attached througha bivalent or trivalent branched linker.
 18. The double-stranded RNAiagent of claim 1, wherein the modifications on the nucleotides areselected from the group consisting of 2′-O-methyl nucleotide,2′-deoxyfluoro nucleotide, 2′-O—N-methylacetamido (2′-O-NMA) nucleotide,a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide,2′-O-aminopropyl (2′-O-AP) nucleotide, 2′-ara-F, and combinationsthereof.
 19. The double-stranded RNAi agent of claim 16, wherein theligand is attached to the 3′ end of the sense strand.
 20. Thedouble-stranded RNAi agent of claim 1, further comprising at least onephosphorothioate or methylphosphonate internucleotide linkage.
 21. Thedouble-stranded RNAi agent of claim 1, wherein the nucleotide at the 1position of the 5′-end of the duplex in the antisense strand is selectedfrom the group consisting of A, dA, dU, U, and dT.
 22. Thedouble-stranded RNAi agent of claim 1, wherein the base pair at the 1position of the 5′-end of the duplex is an AU base pair.
 23. Thedouble-stranded RNAi agent of claim 1, wherein the Y nucleotides containa 2′-fluoro modification.
 24. The double-stranded RNAi agent of claim 1,wherein the Y′ nucleotides contain a 2′-O-methyl modification.
 25. Apharmaceutical composition comprising the double-stranded RNAi agentaccording to claim 1 alone or in combination with a pharmaceuticallyacceptable carrier or excipient.
 26. A method for inhibiting theexpression of a target gene comprising the step of administering thedouble-stranded RNAi agent according to claim 1, in an amount sufficientto inhibit expression of the target gene.
 27. The method of claim 26,wherein the double-stranded RNAi agent is administered throughsubcutaneous or intravenous administration.
 28. A method for deliveringpolynucleotide to specific target in a subject by administering saiddsRNA agent represented by formula (III): sense: 5′n_(p) -N_(a) -(X X X) _(i)-N_(b) -Y Y Y -N_(b) -(Z Z Z)_(j) -N_(a) -n_(q) 3′ antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein: i, j, k, and l are each independently 0 or 1, provided that atleast one of i, j, k, and l is 1; p and q are each independently 0-6;each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 2-20 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides, each N_(b) and N_(b)′independently represents an oligonucleotide sequence comprising 1-10modified nucleotides; each n_(p), n_(p)′, n_(q) and n_(q)′ independentlyrepresents an overhang nucleotide sequence comprising 0-6 nucleotidesequence; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ eachindependently represent one motif of three identical modifications onthree consecutive nucleotides; and wherein the modifications on N_(b) isdifferent than the modification on Y and the modification on N_(b)′ isdifferent than the modification on Y′.
 29. The method of claim 28,wherein said administering step is carried out by an administrationmeans comprising intramuscular, intrabronchial, intrapleural,intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous,cerebrospinal, or combinations thereof.
 30. A method for delivering apolynucleotide to a specific target of a subject, the method comprising:delivering the dsRNA agent according to claim 1 by subcutaneousadministration into the subject, such that the polynucleotide isdelivered into specific target of the subject.